A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

Expression profiling identifies key genes and biological functions associated with eosinophilic esophagitis in human patients.

Introduction: Eosinophilic Esophagitis (EoE) is a chronic allergic disease characterized by progressive inflammation of the esophageal mucosa. This chronic inflammatory disorder affects up to 50 per 100,000 individuals in the United States and Europe yet is limited in treatment options. While the transcriptome of EoE has been reported, few studies have examined the genetics among a cohort including both adult and pediatric EoE populations. To identify potentially overlooked biomarkers in EoE esophageal biopsies that may be promising targets for diagnostic and therapeutic development. Methods: We used microarray analysis to interrogate gene expression using esophageal biopsies from EoE and Control subjects with a wide age distribution. Analysis of differential gene expression (DEGs) and prediction of impaired pathways was compared using conventional transcriptome analysis (TAC) and artificial intelligence-based (ADVAITA) programs. Principal Components Analysis revealed samples cluster by disease status (EoE and Control) irrespective of clinical features like sex, age, and disease severity. Results: Global transcriptomic analysis revealed differential expression of several genes previously reported in EoE (CCL26, CPA3, POSTN, CTSC, ANO1, CRISP3, SPINK7). In addition, we identified differential expression of several genes from the MUC and SPRR families, which have been limited in previous reports. Discussion: Our findings suggest that there is epithelial dysregulation demonstrated by DEGs that may contribute to impaired barrier integrity and loss of epidermal cell differentiation in EoE patients. These findings present two new gene families, SPRR and MUC, that are differentially expressed in both adult and pediatric EoE patients, which presents an opportunity for a future therapeutic target that would be useful in a large demographic of patients.

Successful In Situ Targeting of Pancreatic Tumors in a Novel Orthotopic Porcine Model Using Histotripsy.

OBJECTIVE: New therapeutic strategies and paradigms are direly needed to treat pancreatic cancer. The absence of a suitable pre-clinical animal model of pancreatic cancer is a major limitation to biomedical device and therapeutic development. Traditionally, pigs have proven to be ideal models, especially in the context of designing human-sized instruments, perfecting surgical techniques and optimizing clinical procedures for use in humans. However, pig studies have typically focused on healthy tissue assessments and are limited to general safety evaluations because of the inability to effectively model human tumors. METHODS: Here, we establish an orthotopic porcine model of human pancreatic cancer using RAG2/IL2RG double-knockout immunocompromised pigs and treat the tumors ex vivo and in vivo with histotripsy. RESULTS: Using these animals, we describe the successful engraftment of Panc-1 human pancreatic cancer cell line tumors and characterize their development. To illustrate the utility of these animals for therapeutic development, we determine for the first time, the successful targeting of in situ pancreatic tumors using histotripsy. Treatment with histotripsy resulted in partial ablation in vivo and reduction in collagen content in both in vivo tumor in pig pancreas and ex vivo patient tumor. CONCLUSION: This study presents a first step toward establishing histotripsy as a non-invasive treatment method for pancreatic cancer and exposes some of the challenges of ultrasound guidance for histotripsy ablation in the pancreas. Simultaneously, we introduce a highly robust model of pancreatic cancer in a large mammal model that could be used to evaluate a variety biomedical devices and therapeutic strategies.

Improved Therapeutic Delivery Targeting Clinically Relevant Orthotopic Human Pancreatic Tumors Engrafted in Immunocompromised Pigs Using Ultrasound-Induced Cavitation: A Pilot Study.

Pancreatic tumors can be resistant to drug penetration due to high interstitial fluid pressure, dense stroma, and disarrayed vasculature. Ultrasound-induced cavitation is an emerging technology that may overcome many of these limitations. Low-intensity ultrasound, coupled with co-administered cavitation nuclei consisting of gas-stabilizing sub-micron scale SonoTran Particles, is effective at increasing therapeutic antibody delivery to xenograft flank tumors in mouse models. Here, we sought to evaluate the effectiveness of this approach in situ using a large animal model that mimics human pancreatic cancer patients. Immunocompromised pigs were surgically engrafted with human Panc-1 pancreatic ductal adenocarcinoma (PDAC) tumors in targeted regions of the pancreas. These tumors were found to recapitulate many features of human PDAC tumors. Animals were intravenously injected with the common cancer therapeutics Cetuximab, gemcitabine, and paclitaxel, followed by infusion with SonoTran Particles. Select tumors in each animal were targeted with focused ultrasound to induce cavitation. Cavitation increased the intra-tumor concentrations of Cetuximab, gemcitabine, and paclitaxel by 477%, 148%, and 193%, respectively, compared to tumors that were not targeted with ultrasound in the same animals. Together, these data show that ultrasound-mediated cavitation, when delivered in combination with gas-entrapping particles, improves therapeutic delivery in pancreatic tumors under clinically relevant conditions.

Detecting DNA: An Overview of DNA Recognition by Inflammasomes and Protection against Bacterial Respiratory Infections.

The innate immune system plays a key role in modulating host immune defense during bacterial disease. Upon sensing pathogen-associated molecular patterns (PAMPs), the multi-protein complex known as the inflammasome serves a protective role against bacteria burden through facilitating pathogen clearance and bacteria lysis. This can occur through two mechanisms: (1) the cleavage of pro-inflammatory cytokines IL-1ß/IL-18 and (2) the initiation of inflammatory cell death termed pyroptosis. In recent literature, AIM2-like Receptor (ALR) and Nod-like Receptor (NLR) inflammasome activation has been implicated in host protection following recognition of bacterial DNA. Here, we review current literature synthesizing mechanisms of DNA recognition by inflammasomes during bacterial respiratory disease. This process can occur through direct sensing of DNA or indirectly by sensing pathogen-associated intracellular changes. Additionally, DNA recognition may be assisted through inflammasome-inflammasome interactions, specifically non-canonical inflammasome activation of NLRP3, and crosstalk with the interferon-inducible DNA sensors Stimulator of Interferon Genes (STING) and Z-DNA Binding Protein-1 (ZBP1). Ultimately, bacterial DNA sensing by inflammasomes is highly protective during respiratory disease, emphasizing the importance of inflammasome involvement in the respiratory tract.

Methods to evaluate virus-mediated acute lung inflammation.

As more infectious viruses emerge that result in respiratory illness, there is a significant need to standardize airway harvests and maximize data acquisition. Animal models of respiratory viral infections have been outlined to allow for the analysis of the host immune response and viral pathogenesis kinetics. This chapter outlines two separate tissue harvest protocols following the intranasal infection of mice to investigate both the host immune response and viral pathogenesis. These protocols combine standard laboratory techniques for the analysis of the samples, making it easily integrable for labs without the need for specialized training. In offering two separate yet parallel tissue collection techniques, investigators can ultimately decide which technique will yield the best data for their particular research questions and can maximize data from each animal study.

The Pro-Inflammatory Chemokines CXCL9, CXCL10 and CXCL11 Are Upregulated Following SARS-CoV-2 Infection in an AKT-Dependent Manner.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly transmissible RNA virus that is the causative agent of the Coronavirus disease 2019 (COVID-19) pandemic. Patients with severe COVID-19 may develop acute lung injury (ALI) or acute respiratory distress syndrome (ARDS) and require mechanical ventilation. Key features of SARS-CoV-2 induced pulmonary complications include an overexpression of pro-inflammatory chemokines and cytokines that contribute to a 'cytokine storm.' In the current study an inflammatory state in Calu-3 human lung epithelial cells was characterized in which significantly elevated transcripts of the immunostimulatory chemokines CXCL9, CXCL10, and CXCL11 were present. Additionally, an increase in gene expression of the cytokines IL-6, TNFα, and IFN-γ was observed. The transcription of CXCL9, CXCL10, IL-6, and IFN-γ was also induced in the lungs of human transgenic angiotensin converting enzyme 2 (ACE2) mice infected with SARS-CoV-2. To elucidate cell signaling pathways responsible for chemokine upregulation in SARS-CoV-2 infected cells, small molecule inhibitors targeting key signaling kinases were used. The induction of CXCL9, CXCL10, and CXCL11 gene expression in response to SARS-CoV-2 infection was markedly reduced by treatment with the AKT inhibitor GSK690693. Samples from COVID-19 positive individuals also displayed marked increases in CXCL9, CXCL10, and CXCL11 transcripts as well as transcripts in the AKT pathway. The current study elucidates potential pathway specific targets for reducing the induction of chemokines that may be contributing to SARS-CoV-2 pathogenesis via hyperinflammation.