Microfluidic Synthesis of Scalable Layer-by-Layer Multiple Antigen Nano-Delivery Platform for SARS-CoV-2 Vaccines.

The COVID-19 outbreak was a global pandemic with wide-ranging healthcare implications. Although several mRNA-based vaccines delivered using lipid nanoparticles (LNP) have been approved and demonstrated efficacy at reducing the severity and spread of infection, continued rapid viral evolution and disadvantages currently associated with LNP delivery vehicles (such as toxicity) are driving the design of next-generation SARS-CoV-2 vaccines. Herein, we describe the development of a trimethylated chitosan-based nanoparticle layer-by-layer (LbL) delivery platform for multiple antigens as a scalable and safe COVID-19 vaccine, known as, "LbL-CoV19". These vaccine candidates have been demonstrated to be biocompatible, safe, and effective at stimulating both humoral and cellular responses for protection in preclinical studies. Preliminary results also indicate that LbL-CoV19 can potentially achieve rapid, long-lasting, and broad protection against the SARS-CoV-2 challenge. The "plug-and-play" platform technology is well suited to preparedness for future pandemics and disease outbreaks.

Short Carbon Nanotube-Based Delivery of mRNA for HIV-1 Vaccines.

Developing a safe and effective preventive for HIV-1 remains the hope for controlling the global AIDS epidemic. Recently, mRNA vaccines have emerged as a promising alternative to conventional vaccine approaches, primarily due to their rapid development and potential for low-cost manufacture. Despite the advantages of mRNA vaccines, challenges remain, especially due to the adverse effects of the delivery vehicle and low delivery efficiency. As a result, Luna Labs is developing a short carbon nanotube-based delivery platform (NanoVac) that can co-deliver mRNA and HIV-1 glycoproteins to the immune system efficiently with negligible toxicity. Surface chemistries of NanoVac were optimized to guide antigen/mRNA loading density and presentation. Multiple formulations were engineered for compatibility with both intramuscular and intranasal administration. NanoVac candidates demonstrated immunogenicity in rabbits and generated human-derived humoral and cellular responses in humanized mice (HIS). Briefly, 33% of the HIV-1-infected HIS mice vaccinated with NanoVac-mRNA was cleared of virus infection by 8-weeks post-infection. Finally, NanoVac stabilized the loaded mRNA against degradation under refrigeration for at least three months, reducing the cold chain burden for vaccine deployment.

An immunostimulatory glycolipid that blocks SARS-CoV-2, RSV, and influenza infections in vivo.

Prophylactic vaccines for SARS-CoV-2 have lowered the incidence of severe COVID-19, but emergence of viral variants that are antigenically distinct from the vaccine strains are of concern and additional, broadly acting preventive approaches are desirable. Here, we report on a glycolipid termed 7DW8-5 that exploits the host innate immune system to enable rapid control of viral infections in vivo. This glycolipid binds to CD1d on antigen-presenting cells and thereby stimulates NKT cells to release a cascade of cytokines and chemokines. The intranasal administration of 7DW8-5 prior to virus exposure significantly blocked infection by three different authentic variants of SARS-CoV-2, as well as by respiratory syncytial virus and influenza virus, in mice or hamsters. We also found that this protective antiviral effect is both host-directed and mechanism-specific, requiring both the CD1d molecule and interferon-[Formula: see text]. A chemical compound like 7DW8-5 that is easy to administer and cheap to manufacture may be useful not only in slowing the spread of COVID-19 but also in responding to future pandemics long before vaccines or drugs are developed.

A pancreatic cancer mouse model with human immunity.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a tumor immune microenvironment (TIME) that promotes resistance to immunotherapy. A preclinical model system that facilitates studies of the TIME and its impact on the responsiveness of human PDAC to immunotherapies remains an unmet need. We report a novel mouse model, which develops metastatic human PDAC that becomes infiltrated by human immune cells recapitulating the TIME of human PDAC. The model may serve as a versatile platform to study the nature of human PDAC TIME and its response to various treatments.

Entrance-sealing behavior in the home cage: a defensive response to potential threats linked to the serotonergic system and manifestation of repetitive/stereotypic behavior in mice.

The security of animal habitats, such as burrows and nests, is vital for their survival and essential activities, including eating, mating, and raising offspring. Animals instinctively exhibit defensive behaviors to protect themselves from imminent and potential threats. In 1963, researchers reported wild rats sealing the entrances to their burrows from the inside using materials such as mud, sand, and vegetation. This behavior, known as "entrance sealing (ES)," involves repetitive movements of their nose/mouth and forepaws and is likely a proactive measure against potential intruders, which enhances burrow security. These observations provide important insights into the animals' ability to anticipate potential threats that have not yet occurred and take proactive actions. However, this behavior lacks comprehensive investigation, and the neural mechanisms underpinning it remain unclear. Hypothalamic perifornical neurons expressing urocortin-3 respond to novel objects/potential threats and modulate defensive responses to the objects in mice, including risk assessment and burying. In this study, we further revealed that chemogenetic activation of these neurons elicited ES-like behavior in the home-cage. Furthermore, behavioral changes caused by activating these neurons, including manifestations of ES-like behavior, marble-burying, and risk assessment/burying of a novel object, were effectively suppressed by selective serotonin-reuptake inhibitors. The c-Fos analysis indicated that ES-like behavior was potentially mediated through GABAergic neurons in the lateral septum. These findings underscore the involvement of hypothalamic neurons in the anticipation of potential threats and proactive defense against them. The links of this security system with the manifestation of repetitive/stereotypic behaviors and the serotonergic system provide valuable insights into the mechanisms underlying the symptoms of obsessive-compulsive disorder.

Multiplexed single-cell analysis reveals prognostic and nonprognostic T cell types in human colorectal cancer.

Clinical outcomes in colorectal cancer (CRC) correlate with T cell infiltrates, but the specific contributions of heterogenous T cell types remain unclear. To investigate the diverse function of T cells in CRC, we profiled 37,931 T cells from tumors and adjacent normal colon of 16 patients with CRC with respect to transcriptome, TCR sequence, and cell surface markers. Our analysis identified phenotypically and functionally distinguishable effector T cell types. We employed single-cell gene signatures from these T cell subsets to query the TCGA database to assess their prognostic significance. We found 2 distinct cytotoxic T cell types. GZMK+KLRG1+ cytotoxic T cells were enriched in CRC patients with good outcomes. GNLY+CD103+ cytotoxic T cells with a dysfunctional phenotype were not associated with good outcomes, despite coexpression of CD39 and CD103, markers that denote tumor reactivity. We found 2 distinct Treg subtypes associated with opposite outcomes. While total Tregs were associated with good outcomes, CD38+ Tregs were associated with bad outcomes independently of stage and possessed a highly suppressive phenotype, suggesting that they inhibit antitumor immunity in CRC. These findings highlight the potential utility of these subpopulations in predicting outcomes and support the potential for novel therapies directed at CD38+ Tregs or CD8+CD103+ T cells.

Length of maternal schooling and children's risk of malaria infection: evidence from a natural experiment in Uganda.

Introduction: An estimated 216 million cases of malaria occurred worldwide every year. Cross-sectional studies have reported negative association between maternal education and child malaria risks; however, no randomised trial or quasi-experimental study using a natural experiment has confirmed a causal relationship between these two factors. I used the free primary education reform in Uganda to assess the causal effects of maternal schooling on children's risk of malaria infection. Methods: Malaria biomarkers of children aged <5 years were collected from the 2009 and 2014 Uganda Malaria Indicator Surveys (n=5316). In 1997, the government eliminated tuition requirements in primary schools, which increased the educational attainment of the affected cohorts. Using exposure to the reform as an instrumental variable, I used a two-stage least squares approach to estimate the causal effects of maternal year of education on the probability that a child would contract malaria at the time of the survey. I also evaluated the cost-effectiveness of primary schooling as a malaria control intervention. Results: One extra year of maternal education reduced children's risk of malaria infection by 7.5 percentage points (p=0.057) from baseline (34.9%). The length of maternal education was also positively associated with insecticide-treated bednet usage by their children. The results were robust to a variety of sensitivity tests. Primary schooling for women was a cost-effective intervention to reduce children's malaria infection. Conclusion: Improving access to primary education could be a cost-effective measure to reduce malaria prevalence among children of educated mothers aged <5 years in malaria-endemic countries.

Feedback regulation of Arid5a and Ppar-γ2 maintains adipose tissue homeostasis.

Immune cells infiltrate adipose tissues and provide a framework to regulate energy homeostasis. However, the precise underlying mechanisms and signaling by which the immune system regulates energy homeostasis in metabolic tissues remain poorly understood. Here, we show that the AT-rich interactive domain 5A (Arid5a), a cytokine-induced nucleic acid binding protein, is important for the maintenance of adipose tissue homeostasis. Long-term deficiency of Arid5a in mice results in adult-onset severe obesity. In contrast, transgenic mice overexpressing Arid5a are highly resistant to high-fat diet-induced obesity. Inhibition of Arid5a facilitates the in vitro differentiation of 3T3-L1 cells and fibroblasts to adipocytes, whereas its induction substantially inhibits their differentiation. Molecular studies reveal that Arid5a represses the transcription of peroxisome proliferator activated receptor gamma 2 (Ppar-γ2) due to which, in the absence of Arid5a, Ppar-γ2 is persistently expressed in fibroblasts. This phenomenon is accompanied by enhanced fatty acid uptake in Arid5a-deficient cells, which shifts metabolic homeostasis toward prolipid metabolism. Furthermore, we show that Arid5a and Ppar-γ2 are dynamically counterregulated by each other, hence maintaining adipogenic homeostasis. Thus, we show that Arid5a is an important negative regulator of energy metabolism and can be a potential target for metabolic disorders.

A study of the prebiotic effect of lactulose at low dosages in healthy Japanese women.

To investigate the prebiotic effect of lactulose at low dosages, we assessed changes in defaecation frequency following ingestion of 1, 2, or 3 g/day of lactulose for 2 weeks. Each test was carried out after a 2-week washout period. This was an open-label, before-after trial that enrolled 26 healthy Japanese women. The defaecation frequency, number of defaecation days, and number of faecal bifidobacteria increased significantly compared with before ingestion of 1, 2, and 3 g/day of lactulose. These results suggest that even 1 g/day of lactulose could have a prebiotic effect.

Multiple Micronutrient Supplementation Using Spirulina platensis during the First 1000 Days is Positively Associated with Development in Children under Five Years: A Follow up of A Randomized Trial in Zambia.

Early childhood development relies on various micronutrients. We recently reported that home fortification of complementary foods using spirulina reduced the time to attain motor milestones in Zambian infants. The objective of this study is to estimate the long-term associations between spirulina supplementation during the first 1000 days and child gross motor development, fine motor development, language, and personal⁻social skills at preschool age. We used longitudinal data from a randomized trial conducted in Zambia. In 2015, 501 infants (age, 6⁻18 months) were provided daily supplements of maize-soy-based porridge with spirulina (SP) and without spirulina (CON). Supplementation period lasted for 16 months. In January 2018, children who participated in the initial trial were resurveyed (CON: 182 children; SP: 188 children; now aged 36⁻48 months). We assessed the infants' gross motor development, fine motor development, language, and personal⁻social skills using a modified version of Malawi Development Assessment Tool. The initial clinical trial registration number was NCT03523182. Children in the SP group had higher scores in gross and fine motor development, language, and social skills than those in the CON group. Home fortification of complementary foods using spirulina during the first 1000 days improved development among Zambian children at preschool age.

Multiple micronutrient supplementation using spirulina platensis and infant growth, morbidity, and motor development: Evidence from a randomized trial in Zambia.

In developing countries, micronutrient deficiency in infants is associated with growth faltering, morbidity, and delayed motor development. One of the potentially low-cost and sustainable solutions is to use locally producible food for the home fortification of complementary foods. This study aimed to test the hypothesis that locally producible spirulina platensis supplementation would achieve the following: 1) increase infant physical growth, 2) reduce morbidity, and 3) improve motor development. We randomly assigned 501 Zambian infants into the control group or the spirulina group. Children in the control group (n = 250) received a soya-maize-based porridge for 12 months; those in the spirulina group (n = 251) received the same food with the addition of spirulina. We assessed the change in infants' anthropometric status, morbidity (probable pneumonia, cough, probable malaria, and fever), and motor development over 12 months. The baseline characteristics were not different between the two groups. The attrition rate (47/501) was low. The physical growth of infants in the two groups was similar at 12 months of intervention, as measured by height-for-age z-scores and weight-for-age z-scores. Infants in the spirulina group were 11 percentage points less likely to develop a cough (CI: -0.23, -0.00; P < 0.05) and were more likely to be able to walk alone at 15 months (0.96 ± 0.19) than infants in the control group (0.92 ± 0.28). Home-fortification of complementary foods using spirulina had positive effects on upper respiratory infection morbidity prevention and motor milestone acquisition among Zambian infants.

A Potential Therapeutic Target RNA-binding Protein, Arid5a for the Treatment of Inflammatory Disease Associated with Aberrant Cytokine Expression.

BACKGROUND: Infection, tissue damage and aging can cause inflammation with high levels of inflammatory cytokines. Overproduction of inflammatory cytokines often leads to systemic inflammatory response syndrome (SIRS), severe sepsis, and septic shock. However, prominent therapeutic targets have not been found, although the incidence of sepsis is likely to increase annually. Our recent studies indicate that some RNA-binding proteins, which control gene expression of inflammatory cytokines at the post-transcriptional level, may play a critical role in inflammatory diseases such as sepsis. RESULTS: 1) One of the RNA-binding proteins, AT-rich interactive domain-containing 5a (Arid5a) promotes cytokine production through control of mRNA half-lives of pro-inflammatory molecules such as IL-6, STAT3, T-bet, and OX40 in activated macrophages and T cells. Arid5a KO mice are refractory to endotoxin shock, bleomycininduced lung injury, and inflammatory autoimmune disease. 2) Chlorpromazine (CPZ), which is recognized as a psychotic drug, impairs post-transcriptional gene expression of Il6 in LPS-stimulated macrophages: CPZ inhibits the binding activity of Arid5a to the 3'UTR of Il6 mRNA, thereby destabilizing Il6 mRNA possibly through suppression of Arid5a expression. 3) CPZ has strong suppressive effects on cytokine production such as TNF-α in vivo. Mice with treatment of CPZ are resistant to lipopolysaccharide (LPS)-induced shock. CONCLUSION: Thus, Arid5a contributes to the activation of macrophages and T cells through positive control of mRNA half-lives of inflammatory cytokines and its related molecules, which might lead to cytokine storm. Interestingly, Arid5a was identified from an inhibitory effect of CPZ on IL-6 production in macrophages activated by LPS. Therefore, CPZ derivatives or Arid5a inhibitors may have a potential to suppress severe sepsis through control of post-transcriptional gene expression.

Regulation of inflammatory responses by dynamic subcellular localization of RNA-binding protein Arid5a.

Adenine-thymine (AT)-rich interactive domain 5a (Arid5a) is an RNA-binding protein found in the cytoplasm and nucleus of normally growing cells. Although Arid5a is known to play an important role in immune regulation, whether and how Arid5a subcellular localization impacts immune regulation has remained unclear. In this study, we generated Arid5a transgenic (TG) mice to address this question. While ectopic Arid5a overexpression did not affect expression of inflammatory cytokines under unstimulated conditions, significantly higher levels of inflammatory cytokines, such as IL-6, were produced in response to lipopolysaccharide (LPS) stimulation. Consistent with this, TG mice were more sensitive to LPS treatment than wild-type mice. We also found that Arid5a is imported into the nucleus via a classical importin-α/ß1-mediated pathway. On stimulation, nuclear Arid5a levels were decreased, while there was a concomitant increase in cytoplasmic Arid5a. Arid5a is associated with up-frameshift protein 1, and its nuclear export is regulated by a nuclear export receptor, chromosomal region maintenance 1. Taken together, these data indicate that Arid5a is a dynamic protein that translocates to the cytoplasm from the nucleus so as to properly exert its dual function in mRNA stabilization and transcriptional regulation during inflammatory conditions.

Arid5a stabilizes OX40 mRNA in murine CD4+ T cells by recognizing a stem-loop structure in its 3'UTR.

AT-rich interactive domain-containing protein 5a (Arid5a) is an RNA-binding protein (RBP) required for autoimmunity via stabilization of interleukin-6 (Il6) and signal transducer and activator of transcription 3 (STAT3) mRNAs. However, the roles of Arid5a in Th17 cells and its association with autoimmunity remain unknown. Here, we show that the levels of Arid5a and OX40 are correlated in CD4+ T cells under Th17 conditions in an IL-6-dependent manner. Lack of Arid5a in T cells reduced OX40 expression levels and repressed IL-17 production in response to OX40 ligation. Arid5a stabilized OX40 mRNA by recognizing the alternative decay element (ADE)-like stem-loop (SL) in the 3' untranslated region (3'UTR). Interestingly, Arid5a impaired the RNA-destabilizing functions of Regnase-1 and Roquin-1 on OX40 ADE-like SL. In EAE, Arid5a-deficient mice exhibited resistance to EAE, with reduced OX40 expression in CD4+ T cells, and the number of CD4+ CD45+ T cells was decreased in CNS. Furthermore, ameliorated EAE was induced by adoptive transfer of Arid5a-/- encephalitogenic CD4+ T cells expressing less OX40 mRNA and producing less IL-17. In conclusion, our findings indicate that the Arid5a/OX40 axis in CD4+ T cells may have important implications in pathogenesis of autoimmune diseases such as EAE.

Arid5a-deficient mice are highly resistant to bleomycin-induced lung injury.

Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are among the major causes of death worldwide due to acute inflammation in the lung. AT-rich interactive domain-containing 5a (Arid5a) is an RNA-binding protein involved in inflammatory autoimmune disease through post-transcriptional control of Il6, Stat3 and Tbx21 gene expression. We found that Arid5a-deficient mice were highly refractory to bleomycin (BLM)-induced lethality. Arid5a deficiency suppressed lung pathology, cytokine production (especially, IL-6), and clinical symptoms in BLM-treated mice. Production of reactive oxygen species (ROS) in response to BLM-induced cellular damage was inhibited in Arid5a-deficient mice, potentially affecting the level of oxidized 1-palmitoyl-2-arachidonoyl-phosphaticylcholine (OxPAPC) production. OxPAPC, which is supposed to be a TLR4/TLR2 ligand, stimulated expression of the Arid5a and Il6 genes. Thus, reduction of ROS production in Arid5a-deficient mice could mitigate OxPAPC production, which in turn decreases IL-6 production in vivo due to dysregulated post-transcriptional regulation by loss of Arid5a. Therefore, the control of Arid5a expression represents a potential therapeutic target for treatment of ALI and ARDS.

TLR4-induced NF-κB and MAPK signaling regulate the IL-6 mRNA stabilizing protein Arid5a.

The AT-rich interactive domain-containing protein 5a (Arid5a) plays a critical role in autoimmunity by regulating the half-life of Interleukin-6 (IL-6) mRNA. However, the signaling pathways underlying Arid5a-mediated regulation of IL-6 mRNA stability are largely uncharacterized. Here, we found that during the early phase of lipopolysaccharide (LPS) stimulation, NF-κB and an NF-κB-triggered IL-6-positive feedback loop activate Arid5a gene expression, increasing IL-6 expression via stabilization of the IL-6 mRNA. Subsequently, mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) promotes translocation of AU-rich element RNA-binding protein 1 (AUF-1) from the nucleus to the cytoplasm, where it destabilizes Arid5a mRNA by binding to AU-rich elements in the 3΄ UTR. This results in downregulation of IL-6 mRNA expression. During the late phase of LPS stimulation, p38 MAPK phosphorylates Arid5a and recruits the WW domain containing E3 ubiquitin protein ligase 1 (WWP1) to its complex, which in turn ubiquitinates Arid5a in a K48-linked manner, leading to its degradation. Inhibition of Arid5a phosphorylation and degradation increases production of IL-6 mRNA. Thus, our data demonstrate that LPS-induced NF-κB and MAPK signaling are required to control the regulation of the IL-6 mRNA stabilizing molecule Arid5a. This study therefore substantially increases our understanding of the mechanisms by which IL-6 is regulated.

Regulation of IL-6 in Immunity and Diseases.

Interleukin-6 (IL-6) is a prototypical cytokine with functional pleiotropy and plays an important role in host defense. When infections or tissue injuries occur, IL-6 is promptly produced by monocytes and macrophages and contributes to removal of infectious agents and restoration of damaged tissues through activation of immune, hematological, and acute-phase responses. Once stress is removed from the host, IL-6 synthesis ends, but uncontrolled excessive or persistent IL-6 production plays a pathological role in the development of various inflammatory diseases and cancers, indicating that IL-6 is a double-edged sword for the host. Thus, the proper IL-6 expression is very important for host defense and is strictly controlled by chromatin structure, transcriptional regulation, and posttranscriptional modification. Differentiation status of cells, various transcription factors, RNA-binding proteins, and microRNAs are involved in this process. Since it is assumed that dysregulation of any of these regulatory molecules may cause abnormal IL-6 expression in a particular disease, further elucidation of the factors and processes involved in IL-6 expression can be expected to facilitate to clarification of pathogenesis and to identification of novel target molecule(s) for specific diseases.

Arid5a exacerbates IFN-γ-mediated septic shock by stabilizing T-bet mRNA.

Adenine-thymine (AT)-rich interactive domain containing protein 5a (Arid5a) is an RNA-binding protein that has been shown to play an important immune regulatory function via the stabilization of IL-6 and STAT3 mRNA. However, the role of Arid5a in the overwhelming and uncontrolled immune response that leads to septic shock is unknown. Here, we report that Arid5a-deficient mice are highly resistant to lipopolysaccharide (LPS)-induced endotoxic shock and secrete lower levels of major proinflammatory cytokines, including IFN-γ, IL-6, and TNF-α, than WT mice in response to LPS. Arid5a deficiency resulted in decreased levels of IFN-γ under Th1 cell conditions, in which T-box expressed in T cells (T-bet) mRNA expression was inhibited. Arid5a bound to the conserved stem loop structure of the 3'UTR of T-bet and stabilized its mRNA. Arid5a-deficient mice were also resistant to Propionibacterium acnes-primed LPS injection, which is considered to be a T-cell-mediated IFN-γ dependent endotoxic shock mouse model. Thus, regulation of IFN-γ by Arid5a via the stabilization of T-bet mRNA in Th1 cells contributes to the development of septic shock in mice. In addition, our previous study suggests that Arid5a control the IL-6 level in vivo in response to LPS by stabilization of IL-6 mRNA. We also observed that neutralization of IFN-γ and IL-6 significantly recovered the mice from endotoxic shock. Taken together, we conclude that Arid5a regulates the augmentation of IL-6 and IFN-γ in response to LPS, which possibly works synergistically for amplification of various other cytokines that ultimately cause the development of septic shock in mice.

CD5: A New Partner for IL-6.

IL-6-STAT3 axis is known as a key factor of tumor progression. In this issue of Immunity, Yu and colleagues (2016) show that IL-6-binding CD5, rather than IL-6 receptor-α in B cells, amplifies STAT3 activation via JAK-STAT signaling to promote cancer.

Arid5a regulates naive CD4+ T cell fate through selective stabilization of Stat3 mRNA.

Balance in signal transducer and activator of transcription (STAT) activation is a key factor in regulating the fate of naive CD4(+)T cells. Here, we demonstrate that AT-rich interactive domain-containing protein 5a (Arid5a) in T cells directs naive CD4(+)T cells to differentiate into inflammatory CD4(+)T cells, especially Th17 cells, through selective stabilization of Stat3(but not Stat1 and Stat5) mRNA in an IL-6-dependent manner. Loss of Arid5a in T cells led to reduction of STAT3 level under Th17-polarizing conditions, whereas STAT1 and STAT5 in Arid5a-deficient T cells were highly activated compared with those of WT T cells under the same conditions. These cells displayed the feature of antiinflammatory (Il10-expressing) CD4(+)T cells. Thus, we show a T cell-intrinsic role of Arid5a on fate decisions of naive CD4(+)T cells through selective stabilization of Stat3 mRNA.