Single-Cell Sequencing of Peripheral Mononuclear Cells Reveals Distinct Immune Response Landscapes of COVID-19 and Influenza Patients.

The coronavirus disease 2019 (COVID-19) pandemic poses a current world-wide public health threat. However, little is known about its hallmarks compared to other infectious diseases. Here, we report the single-cell transcriptional landscape of longitudinally collected peripheral blood mononuclear cells (PBMCs) in both COVID-19- and influenza A virus (IAV)-infected patients. We observed increase of plasma cells in both COVID-19 and IAV patients and XIAP associated factor 1 (XAF1)-, tumor necrosis factor (TNF)-, and FAS-induced T cell apoptosis in COVID-19 patients. Further analyses revealed distinct signaling pathways activated in COVID-19 (STAT1 and IRF3) versus IAV (STAT3 and NFκB) patients and substantial differences in the expression of key factors. These factors include relatively increase of interleukin (IL)6R and IL6ST expression in COVID-19 patients but similarly increased IL-6 concentrations compared to IAV patients, supporting the clinical observations of increased proinflammatory cytokines in COVID-19 patients. Thus, we provide the landscape of PBMCs and unveil distinct immune response pathways in COVID-19 and IAV patients.

The breadth of concomitant virological features in a family cluster outbreak of COVID-19 pneumonia.

An outbreak of a novel coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) had emerged in 2019 and rapidly posed a global epidemic. Here, we report the breadth of concomitant virological features of a family cluster with COVID-19. The period of virus shedding is significantly different between upper respiratory and feces samples. Even the SARS-CoV-2 virus titers were undetectable in feces, it could be positive again soon and likely related to fluctuated inflammation levels (interleukin-6, etc.) and lowered immune responses (CD4 + T lymphocyte, etc.). Our findings expand the novel understanding of the breadth of concomitant virological features during a non-severe family cluster of COVID-19.

A recombinant influenza virus with a CTLA4-specific scFv inhibits tumor growth in a mouse model.

Oncolytic viruses (OV) have shown excellent safety and efficacy in preclinical and clinical studies. Influenza A virus (IAV) is considered a promising oncolytic virus. In this report, we generated a recombinant influenza virus expressing an immune checkpoint blockade agent targeting CTLA4. Using reverse genetics, a recombinant influenza virus, termed rFlu-CTLA4, encoding the heavy chain of a CTLA4 antibody on the PB1 segment and the light chain of the CTLA4 antibody on the PA segment was produced. RFlu-CTLA4 could replicate to high titers, and antibodies were produced in the allantoic fluid of infected eggs. Furthermore, the selective cytotoxicity of the virus was higher in various hepatocellular carcinoma cancer cell lines than in the normal cell line L02 in vitro, as indicated by MTS assays. More importantly, in a subcutaneous H22 mouse hepatocarcinoma model, intratumoral injections of rFlu-CTLA4 inhibited the growth of treated tumors and increased the overall survival of mice compared with injections of the PR8 virus. Taken together, these results warrant further exploration of this novel recombinant influenza virus for its potential use as a single or combination agent for cancer immunotherapy.

Long noncoding RNA FAM66C promotes tumor progression and glycolysis in intrahepatic cholangiocarcinoma by regulating hsa-miR-23b-3p/KCND2 axis.

Long noncoding RNAs (lncRNAs) are known to be the important regulators in cancer progression. However, the role of lncRNA FAM66C (FAM66C) is yet to be investigated in intrahepatic cholangiocarcinoma (ICC). This study aimed to investigate the effects and related mechanisms of FAM66C in ICC. Human ICC tissues and cell lines were collected. The expression levels of FAM66C, hsa-miR-23b-3p (miR-23b-3p), and KCND2 were detected by qRT-RCR. The transfection experiments were employed to measure the effect of FAM66C on cell viabilities, migration, and invasion in ICC cells by CCK-8, transwell assays. Glycolysis was investigated by glucose consumption, lactate production and ATP levels. The dual-luciferase reporter and RNA pull down assays were conducted as a means of confirming the interactions between FAM66C, miR-23b-3p, and KCND2. Furthermore, the levels of the EMT-associated proteins (KCND2, GLUT1, PKM2, and LDHA) in ICC cells were detected by western blot. FAM66C was increased in ICC tissues and cells, increased cell viability, glycolysis, migration and invasion, and decreased apoptosis were shown in FAM66C overexpressing cells. Mechanistic analyses revealed that FAM66C regulated the downstream target gene KCND2 by sponging miR-23b-3p. FAM66C effect on ICC was further validated in murine xenograft assays. FAM66C knockdown cells gave rise to tumors that were smaller in size, consistent with the role of FAM66C as a promoter of in vivo tumor growth. These data revealed that FAM66C was able to drive ICC tumor progression and glycolytic activity via the miR-23b-3p/KCND2 axis, indicating FAM66C may be a viable target for treating ICC.

SET domain containing 1B gene is mutated in primary hepatic neuroendocrine tumors.

Primary hepatic neuroendocrine tumors (PHNETs) are extremely rare NETs originating from the liver. These tumors are associated with heterogeneous prognosis, and few treatment targets for PHNETs have been identified. Because the major genetic alterations in PHNET are still largely unknown, we performed whole-exome sequencing of 22 paired tissues from PHNET patients and identified 22 recurring mutations of somatic genes involved in the following activities: epigenetic modification (BPTF, MECP2 and WDR5), cell cycle (TP53, ATM, MED12, DIDO1 and ATAD5) and neural development (UBR4, MEN1, GLUL and GIGYF2). Here, we show that TP53 and the SET domain containing the 1B gene (SETD1B) are the most frequently mutated genes in this set of samples (3/22 subjects, 13.6%). A biological analysis suggests that one of the three SETD1B mutants, A1054del, promotes cell proliferation, migration and invasion compared to wild-type SETD1B. Our work unveils that SETD1B A1054del mutant is functional in PHNET and implicates genes including TP53 in the disease. Our findings thus characterize the mutational landscapes of PHNET and implicate novel gene mutations linked to PHNET pathogenesis and potential therapeutic targets.

Long Stress Induced Non-Coding Transcripts 5 (LSINCT5) Promotes Hepatocellular Carcinoma Progression Through Interaction with High-Mobility Group AT-hook 2 and MiR-4516.

BACKGROUND Long non-coding RNAs (lncRNAs) have been implicated in various human cancer types. However, the underlying mechanisms involved in hepatocellular carcinoma (HCC) progression remain poorly understood. MATERIAL AND METHODS In this study, lncRNA array was used to identify HCC related lncRNAs. RNA immunoprecipitation (RIP) followed mass spectrometry was used to explore lncRNA binding proteins. Western blot, quantitative PCR, tumor sphere formation, migration and viability assay were performed to evaluate the oncogenic role of lncRNAs. RESULTS We identified a novel lncRNA named long stress induced non-coding transcripts 5 (LSINCT5) which facilitates HCC progression. LSINCT5 was significantly upregulated in both HCC specimens and cell lines and correlates with poor survival. In vitro experiments showed that LSINCT5 promoted migration and viability of HepG2 and Huh7 cells. The in vivo xenograft mouse model also confirmed an oncogenic role for LSINCT5. RIP in combination with mass spectrometry identified HMGA2 as the LSINCT5 binding partner. LSINCT5 could bind to HMGA2 and decrease proteasome-mediated HMGA2 degradation leading to EMT activation. LSINCT5 also served as a competing endogenous RNA (ceRNA) for miR-4516, resulting in increased STAT3/BclxL expression and attenuated apoptosis. CONCLUSIONS Our data have collectively established a lncRNA LSINCT5 mediated process during HCC carcinogenesis and might have provided novel insight into therapeutic targeting.

An Engineered Influenza a Virus Expressing the Co-Stimulator OX40L as an Oncolytic Agent Against Hepatocellular Carcinoma.

Background: Oncolytic virus (OV) therapy has emerged as a promising novel form of immunotherapy. Moreover, an increasing number of studies have shown that the therapeutic efficacy of OV can be further improved by arming OVs with immune-stimulating molecules. Methods: In this study, we used reverse genetics to produce a novel influenza A virus, termed IAV-OX40L, which contained the immune-stimulating molecule OX40L gene in the influenza virus nonstructural (NS1) protein gene. The oncolytic effect of IAV-OX40L was explored on hepatocellular carcinoma (HCC)HCC cells in vitro and in vivo. Results: Hemagglutination titers of the IAV-OX40L virus were stably 27-28 in specific-pathogen-free chicken embryos. The morphology and size distribution of IAV-OX40L are similar to those of the wild-type influenza. Expression of OX40L protein was confirmed by Western blot and immunofluorescence. MTS assays showed that the cytotoxicity of IAV-OX40L was higher in HCC cells (HepG2 and Huh7) than in normal liver cells (MIHA) in a time- and dose-dependent manner in vitro. We found that intratumoral injection of IAV-OX40L reduced tumor growth and increased the survival rate of mice compared with PR8-treated controls in vivo. In addition, the pathological results showed that IAV-OX40L selectively destroyed tumor tissues without harming liver and lung tissues. CD4+ and CD8+ T cells of the IAV-OX40L group were significantly increased in the splenic lymphocytes of mice. Further validation confirmed that IAV-OX40L enhanced the immune response mainly by activating Th1-dominant immune cells, releasing interferon-γ and interleukin-2. Conclusion: Taken together, our findings demonstrate the novel chimeric influenza OV could provide a potential therapeutic strategy for combating HCC and improve the effectiveness of virotherapy for cancer therapy.

A Recombinant Oncolytic Influenza Virus Carrying GV1001 Triggers an Antitumor Immune Response.

Oncolytic viruses are able to lyse tumor cells selectively in the liver without killing normal hepatocytes, in addition to activating the immune response. Oncolytic virus therapy is expected to revolutionize the treatment of liver cancer, including hepatocellular carcinoma (HCC), one of the most frequent and fatal malignancies. In this study, reverse genetics techniques were exploited to load NA fragments of the A/PuertoRico/8/34 virus (PR8) with GV1001 peptides derived from human telomerase reverse transcriptase. An in vitro assessment of the therapeutic effect of the recombinant oncolytic virus was followed by an in vivo study in mice with HCC. The recombinant virus was verified by sequencing of the recombinant viral gene sequence, and viral virulence was detected by hemagglutination assays and based on the 50% tissue culture infectious dose (TCID50). The morphological structure of the virus was observed by electron microscopy, and GV1001 peptide was localized by cellular immunofluorescence. The selective cytotoxicity of the recombinant oncolytic virus in vitro was demonstrated in cultured HCC cells and normal hepatocytes, as only the tumor cells were killed; the normal cells were not significantly altered. Consistent with the in vitro results, the recombinant oncolytic influenza virus significantly inhibited liver tumor growth in mice in vivo, in addition to inducing an antitumor immune response, including an increase in the number of CD4+ and CD8+ T lymphocytes and, in turn, improving survival. Our results suggest that oncolytic influenza virus carrying GV1001 is a promising immunotherapy in patients with HCC.

Serum Exosome-Derived microRNA-193a-5p and miR-381-3p Regulate Adenosine 5'-Monophosphate-Activated Protein Kinase/Transforming Growth Factor Beta/Smad2/3 Signaling Pathway and Promote Fibrogenesis.

INTRODUCTION: Liver fibrosis results from chronic liver injury and inflammation, often leading to cirrhosis, liver failure, portal hypertension, and hepatocellular carcinoma. Progress has been made in understanding the molecular mechanisms underlying hepatic fibrosis; however, translating this knowledge into effective therapies for disease regression remains a challenge, with considerably few interventions having entered clinical validation. The roles of exosomes during fibrogenesis and their potential as a therapeutic approach for reversing fibrosis have gained significant interest. This study aimed to investigate the association between microRNAs (miRNAs) derived from serum exosomes and liver fibrosis and to evaluate the effect of serum exosomes on fibrogenesis and fibrosis reversal, while identifying the underlying mechanism. METHODS: Using serum samples collected from healthy adults and paired histologic patients with advanced fibrosis or cirrhosis, we extracted human serum exosomes by ultrahigh-speed centrifugation. Transcriptomic analysis was conducted to identify dysregulated exosome-derived miRNAs. Liver fibrosis-related molecules were determined by qRT-PCR, Western blot, Masson staining, and immunohistochemical staining. In addition, we analyzed the importance of serum exosome-derived miRNA expression levels in 42 patients with advanced fibrosis or cirrhosis. RESULTS: Exosome-derived miR-193a-5p and miR-381-3p were associated with fibrogenesis, as determined by transcriptomic screening. Compared with healthy control group, the high expression of serum exosome-derived miR-193a-5p and miR-381-3 in chronic hepatitis B (n = 42) was closely associated with advanced liver fibrosis and cirrhosis. In vitro , exosome-derived miRNA-193a-5p and miR-381-3p upregulated the expression of α-smooth muscle actin, collagen 1a1, and tissue inhibitors of metalloproteinase 1 in the human hepatic stellate cell line at both mRNA and protein levels. DISCUSSION: Serum exosome-derived miR-193a-5p and miR-381-3p regulated the adenosine 5'-monophosphate-activated protein kinase/transforming growth factor beta/Smad2/3 signaling pathway and promoted fibrogenesis.

Enhanced NK cell activation via eEF2K-mediated potentiation of the cGAS-STING pathway in hepatocellular carcinoma.

BACKGROUND: Liver cancer, particularly hepatocellular carcinoma (HCC), is characterized by a high mortality rate, attributed primarily to the establishment of an immunosuppressive microenvironment. Within this context, we aimed to elucidate the pivotal role of eukaryotic elongation factor 2 kinase (eEF2K) in orchestrating the infiltration and activation of natural killer (NK) cells within the HCC tumor microenvironment. By shedding light on the immunomodulatory mechanisms at play, our findings should clarify HCC pathogenesis and help identify potential therapeutic intervention venues. METHODS: We performed a comprehensive bioinformatics analysis to determine the functions of eEF2K in the context of HCC. We initially used paired tumor and adjacent normal tissue samples from patients with HCC to measure eEF2K expression and its correlation with prognosis. Subsequently, we enrolled a cohort of patients with HCC undergoing immunotherapy to examine the ability of eEF2K to predict treatment efficacy. To delve deeper into the mechanistic aspects, we established an eEF2K-knockout cell line using CRISPR/Cas9 gene editing. This step was crucial for verifying activation of the cGAS-STING pathway and the subsequent secretion of cytokines. To further elucidate the role of eEF2K in NK cell function, we applied siRNA-based techniques to effectively suppress eEF2K expression in vitro. For in vivo validation, we developed a tumor-bearing mouse model that enabled us to compare the infiltration and activation of NK cells within the tumor microenvironment following various treatment strategies. RESULTS: We detected elevated eEF2K expression within HCC tissues, and this was correlated with an unfavorable prognosis (30.84 vs. 20.99 months, P = 0.033). In addition, co-culturing eEF2K-knockout HepG2 cells with dendritic cells led to activation of the cGAS-STING pathway and a subsequent increase in the secretion of IL-2 and CXCL9. Moreover, inhibiting eEF2K resulted in notable NK cell proliferation along with apoptosis reduction. Remarkably, after combining NH125 and PD-1 treatments, we found a significant increase in NK cell infiltration within HCC tumors in our murine model. Our flow cytometry analysis revealed reduced NKG2A expression and elevated NKG2D expression and secretion of granzyme B, TNF-α, and IFN-γ in NK cells. Immunohistochemical examination confirmed no evidence of damage to vital organs in the mice treated with the combination therapy. Additionally, we noted higher levels of glutathione peroxidase and lipid peroxidation in the peripheral blood serum of the treated mice. CONCLUSION: Targeted eEF2K blockade may result in cGAS-STING pathway activation, leading to enhanced infiltration and activity of NK cells within HCC tumors. The synergistic effect achieved by combining an eEF2K inhibitor with PD-1 antibody therapy represents a novel and promising approach for the treatment of HCC.

Three-dimensional chromatin landscapes in hepatocellular carcinoma associated with hepatitis B virus.

BACKGROUND: Three-dimensional (3D) chromatin architecture frequently altered in cancer. However, its changes during the pathogenesis of hepatocellular carcinoma (HCC) remained elusive. METHODS: Hi-C and RNA-seq were applied to study the 3D chromatin landscapes and gene expression of HCC and ANHT. Hi-C Pro was used to generate genome-wide raw interaction matrices, which were normalized via iterative correction (ICE). Moreover, the chromosomes were divided into different compartments according to the first principal component (E1). Furthermore, topologically associated domains (TADs) were visualized via WashU Epigenome Browser. Furthermore, differential expression analysis of ANHT and HCC was performed using the DESeq2 R package. Additionally, dysregulated genes associated with 3D genome architecture altered were confirmed using TCGA, qRT-PCR, immunohistochemistry (IHC), etc. RESULTS: First, the intrachromosomal interactions of chr1, chr2, chr5, and chr11 were significantly different, and the interchromosomal interactions of chr4-chr10, chr13-chr21, chr15-chr22, and chr16-chr19 are remarkably different between ANHT and HCC, which resulted in the up-regulation of TP53I3 and ZNF738 and the down-regulation of APOC3 and APOA5 in HCC. Second, 49 compartment regions on 18 chromosomes have significantly switched (A-B or B-A) during HCC tumorigenesis, contributing to up-regulation of RAP2A. Finally, a tumor-specific TAD boundary located on chr5: 6271000-6478000 and enhancer hijacking were identified in HCC tissues, potentially associated with the elevated expression of MED10, whose expression were associated with poor prognosis of HCC patients. CONCLUSION: This study demonstrates the crucial role of chromosomal structure variation in HCC oncogenesis and potential novel biomarkers of HCC, laying a foundation for cancer precision medicine development.

Protective efficacy of a novel recombinant influenza virus carrying partial human metapneumovirus F protein epitopes.

Human metapneumovirus (HMPV) is a leading cause of acute respiratory tract infections in infants and children. Currently, no approved HMPV vaccine is available. We developed a novel recombinant influenza virus, which carried partial HMPV F protein (HMPV-F) epitopes, utilizing reverse genetics. The novel single-stranded RNA virus, termed rFLU-HMPV/F-NA, was synthesized in the neuraminidase (NA) fragment of influenza virus A/PuertoRico/8/34 (PR8). The morphological characteristics of rFLU-HMPV/F-NA were consistent with the wild-type flu virus. The virus could passage in specific pathogen-free (SPF) chicken embryos for at least five consecutive generations with haemagglutinin (HA) titres of 28-9 or 8-9LogTCID50/mL. BALB/c mice were intranasally immunized at 21-day intervals with 104 TCID50 (low-dose group) or 106 TCID50 (high-dose group) rFLU-HMPV/F-NA, and PBS or PR8 vaccine was used for the control group. rFLU-HMPV/F-NA induced robust humoral, mucosal, and cellular immune responses in vivo in a dose-dependent manner. More importantly, wt clinical HMPV isolate challenge studies showed that rFLU-HMPV/F-NA provided significant immune protection against HMPV infection compared to the PBS or PR8 vaccine control group, as shown by improved histopathological changes and reduced viral titres in the lungs of immunized mice post-challenge. These findings demonstrate that rFLU-HMPV/F-NA has potential as a promising HMPV candidate vaccine and warrants further investigation into its control of HMPV infection.

Decreased granzyme-B expression in CD11c+CD8+ T cells associated with disease progression in patients with HBV-related hepatocellular carcinoma.

Introduction: CD11c+CD8+ T cells are an unconventional CD8+ T cell subset that exerts antiviral activity in infectious diseases. However, its characteristics in hepatocellular carcinoma (HCC) have not been elucidated. Methods: Twenty-six patients with hepatitis B virus (HBV)-related HCC and 25 healthy controls (HC) were enrolled. The frequency and phenotype of CD11c+CD8+ T cells in peripheral blood and tumors in situ were detected by flow cytometry and immunohistochemistry. Results: Both the HCC group and HC group had similar frequency and phenotype characteristics of CD11c+CD8+ T cells in the periphery. CD11c+CD8+ T cells were mainly composed of effector T cells, most of which were CD45RA+CCR7-. Compared with CD11c-CD8+ T cells, CD11c+CD8+ T cells had a higher proportion of CD38 and HLA-DR double positive, and expressed high levels of granzyme-B (GB) and degranulation marker CD107a, and produced high levels of interleukin-2 (IL-2), tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ). However, the ability of degranulation and TNF-α production of CD11c+CD8+ T cells in patients with HCC were significantly lower than that in healthy controls. The GB expression level of peripheral CD11c+CD8+ T cells in patients with advanced stage of HCC was significantly lower than that in patients with early stage of HCC, and the GB expression level of liver-infiltrating CD11c+CD8+ T cells in tumor tissues was lower than that in non-tumor tissues. More importantly, the GB expression level of peripheral CD11c+CD8+ T cells was negatively correlated with tumor volume. Conclusions: These findings indicate that CD11c+CD8+ T cells may have potential anti-tumor activity and that GB+CD11c+CD8+ T cells are associated with disease progression in patients with HBV-related HCC.

Identification of a new five-gene risk score for risk stratification and prognosis prediction in HCC.

Approximately 80% of primary liver cancer (PLC) is hepatocellular carcinoma (HCC), and the prognosis of HCC patients is unfavorable. Further studies are required to develop new prognostic tools for predicting the HCC patients' prognosis. The univariate Cox and LASSO regression were utilized to develop the multi-gene risk score. Single-sample Gene Set Enrichment Analysis (ssGSEA) was employed to assess differences of immune functions and cells. The model performance was evaluated by calibration curve and receiver operating characteristic curve (ROC). And qRT-PCR was utilized to evaluate the genes expression in clinical samples. Finally, a novel five-gene (KIF20A, CENPA, HMMR, G6PD, and ADH4) risk score was developed. Based on the median value of patients' risk scores, patients were divided into two groups: high-risk group and low-risk group. The Overall survival (OS) of patients in high-risk group was obviously poorer than that in the low-risk group. And the five-gene risk score was an independent risk factor correlated with patients' OS. Besides, a nomogram consisting of TNM stage and risk score was established. The results of decision curve, calibration curve, and ROC presented that the prognostic risk score and the nomogram had great predictive capability. Besides, ADH4's mRNA was reduced in HCC tissues, while the mRNA of KIF20A, CENPA, HMMR, and G6PD were overexpressed in HCC tissues. We developed a novel five-gene risk score that could predict HCC patients' prognosis. And these five genes could be promising therapeutic targets for HCC. The five-gene risk score and nomogram may be useful prognostic tools for HCC.

Intestinal Candida albicans Promotes Hepatocarcinogenesis by Up-Regulating NLRP6.

Hepatocellular carcinoma (HCC), a primary liver cancer, is closely associated with the gut microbiota. However, the role of gut fungi in the development of HCC remains unclear. The aim of this study was to explore the influence of intestinal Candida albicans on HCC. Here, We found that patients with HCC showed significantly decreased diversity of the gut mycobiome and increased abundance of C. albicans, compared to the patients with liver cirrhosis. The gavage of C. albicans in the WT models increased the tumor size and weight and influenced the plasma metabolome, which was indicated by alterations in 117 metabolites, such as L-carnitine and L-acetylcarnitine, and several KEGG enriched pathways, such as phenylalanine metabolism and citrate cycle. Moreover, the expression of nucleotide oligomerization domain-like receptor family pyrin domain containing 6 (NLRP6) in the intestinal tissues and primary intestinal epithelial cells of the WT mice interacted with C. albicans increased. Notably, the colonization of C. albicans had no effect on tumor growth in Nlrp6 -/- mice. In conclusion, the abnormal colonization of C. albicans reprogrammed HCC metabolism and contributed to the progression of HCC dependent on NLRP6, which provided new targets for the treatment of HCC.

Integrative immunogenomic analysis reveals transcriptional and immune-related differences in hepatocellular carcinoma patients with different disease-free survival.

A comprehensive investigation of the neoantigen spectrum and immune infiltration in patients with hepatocellular carcinoma (HCC) is lacking. This study aimed to examine the molecular features correlating with better prognoses in HCC patients. 27 paired tumor and normal tissues from 27 HCC patients were collected and performed with whole-exome sequencing. The most frequently mutated gene in 27 HCC patients was TP53 (16/27, 59.26%). Based on the whole median disease-free survival (DFS), all patients were divided into 'long-term' (n = 14, median DFS = 318 weeks) and 'short-term' (n = 13, median DFS = 11 weeks) groups. RNA-seq was performed to compare differentially expressed genes, immune infiltration, and neoantigens. Immunohistochemistry was performed to evaluate the immune infiltration. There were no significant differences in tumor mutation burden, immune score, cytolytic activity score, or neoantigen load between two groups. Compared with the long-term group, significantly increased B lineage (P = 0.0463), myeloid dendritic cells (P = 0.0152), and fibroblast (P = 0.0244) infiltration levels were observed in the short-term group, in which genes involved in ribosome, proteasome, and ECM-receptor interaction pathways were also overexpressed. Additionally, 16 patients with tumor thrombus were explored to identify specific biomarkers for prognosis. We found that patients with tumor thrombus carrying TP53/ARID2 neoantigens had significantly longer DFS. In conclusion, higher B lineage, myeloid dendritic cells, and fibroblast infiltration levels might cause poor prognosis in the short-term group, which also showed higher expression of genes involved in ribosome, proteasome, and ECM-receptor interaction pathways. In patients with tumor thrombus, specific TP53/ARID2 neoantigens may be used as biomarkers toward personalized immunotherapy.

Oncolytic Activity of a Chimeric Influenza A Virus Carrying a Human CTLA4 Antibody in Hepatocellular Carcinoma.

Oncolytic virotherapy belongs to a kind of active immunotherapy, which could trigger a potent antitumor immune response, showing great potential in clinical application. OVs could induce immune responses through the dual mechanisms of selective tumor killing without destroying normal tissues and induction of systemic antitumor immunity. In this study, we successfully rescued a chimeric oncolytic influenza virus carrying a human CTLA4 antibody in the background of the A/PR/8/34 (PR8) virus. The chimeric virus, called rFlu-huCTLA4, contained the heavy and light chains of the human CTLA4 antibody in the PB1 and PA segments of the PR8 virus, respectively. The first-generation hemagglutination (HA) titers of the rFlu-huCTLA4 virus ranged from 27 to 28, which could be passaged stably in specific pathogen-free (SPF) chicken embryos from P1 to P5. The morphology and size distribution of the chimeric virus were consistent with those of the wt influenza virus. The rFlu-huCTLA4 virus could effectively replicate in various cells in time- and dose-dependent manners. ELISA assay revealed that the secreted huCTLA4 antibody levels in chicken embryos increased gradually over time. Furthermore, MTS and crystal violet analysis showed that the selective cytotoxicity of the virus was higher in hepatocellular carcinoma cells (HepG2 and Huh7) than in normal liver cells (MIHA). In vivo experiments displayed that intratumoral injection with rFlu-huCTLA4 reduced tumor growth and increased the survival of mice compared with the PR8 group. More importantly, in the rFlu-huCTLA4 group, we found that CD4+ and CD8 +T cells were significantly increased in tumor-bearing BALB/c mice. Taken together, these findings demonstrated that the chimeric oncolytic virus rFlu-huCTLA4 could selectively destroy hepatocellular carcinoma cells in vitro and in vivo and may provide a promising clinical strategy for targeted immunotherapy of HCC with the oncolytic flu virus.

Therapeutic Efficacy of an Oncolytic Influenza Virus Carrying an Antibody Against Programmed Cell Death 1 in Hepatocellular Carcinoma.

Oncolytic virus therapy is a promising novel immunotherapy. In this report, we engineered a novel oncolytic influenza virus (IV) carrying an antihuman programmed cell death 1 (PD-1) monoclonal antibody utilizing reverse genetics. A reassortant chimeric IV, named rFlu-huPD1, was synthesized as follows: the heavy chain of the PD-1 antibody was encoded on the PB1 fragment, and the light chain of the PD-1 antibody was encoded on the polymerase acid protein fragment. rFlu-huPD1 antibodies were produced in infected ovalantoic eggs and could replicate to high titers. Moreover, selective cytotoxicity of rFlu-huPD1 was upregulated in multiple hepatocellular carcinoma (HCC) cell lines compared with a control, as determined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the activation of T cells in the spleen of tumor-bearing BALB/c mice treated with rFlu-huPD1 was observed, especially cytotoxic CD8+ T cell activation in vivo. In addition, in a patient-derived xenograft liver cancer mouse model, tumor growth was reduced and the overall survival of the mice was increased by intratumoral injections with rFlu-huPD1 compared with wild-type PR8 virus. Taken together, these findings provide evidence for the utility of a combination of oncolytic IVs expressing PD-1 inhibitors for use in HCC virotherapy.

Long non-coding ribonucleic acid W5 inhibits progression and predicts favorable prognosis in hepatocellular carcinoma.

BACKGROUND: Accumulating evidence has revealed that several long non-coding ribonucleic acids (lncRNAs) are crucial in the progress of hepatocellular carcinoma (HCC). AIM: To classify a long non-coding RNA, i.e., lncRNA W5, and to determine the clinical significance and potential roles of lncRNA W5 in HCC. METHODS: The results showed that lncRNA W5 expression was significantly downregulated in HCC cell lines and tissues. Analysis of the association between lncRNA W5 expression levels and clinicopathological features suggested that low lncRNA W5 expression was related to large tumor size (P < 0.01), poor histological grade (P < 0.05) and serious portal vein tumor thrombosis (P < 0.05). Furthermore, Kaplan-Meier survival analysis showed that low expression of lncRNA W5 predicts poor overall survival (P = 0.016). RESULTS: Gain-of-loss function experiments, including cell counting kit8 assays, colony formation assays, and transwell assays, were performed in vitro to investigate the biological roles of lncRNA W5. In vitro experiments showed that ectopic overexpression of lncRNA W5 suppressed HCC cell proliferation, migration and invasion; conversely, silencing of lncRNA W5 promoted cell proliferation, migration and invasion. In addition, acting as a tumor suppressor gene in HCC, lncRNA W5 inhibited the growth of HCC xenograft tumors in vivo. CONCLUSION: These results showed that lncRNA W5 is down-regulated in HCC, and it may suppress HCC progression and predict poor clinical outcomes in patients with HCC. LncRNA W5 may serve as a potential HCC prognostic biomarker in addition to a therapeutic target.

Integrated gut virome and bacteriome dynamics in COVID-19 patients.

SARS-CoV-2 is the cause of the current global pandemic of COVID-19; this virus infects multiple organs, such as the lungs and gastrointestinal tract. The microbiome in these organs, including the bacteriome and virome, responds to infection and might also influence disease progression and treatment outcome. In a cohort of 13 COVID-19 patients in Beijing, China, we observed that the gut virome and bacteriome in the COVID-19 patients were notably different from those of five healthy controls. We identified a bacterial dysbiosis signature by observing reduced diversity and viral shifts in patients, and among the patients, the bacterial/viral compositions were different between patients of different severities, although these differences are not entirely distinguishable from the effect of antibiotics. Severe cases of COVID-19 exhibited a greater abundance of opportunistic pathogens but were depleted for butyrate-producing groups of bacteria compared with mild to moderate cases. We replicated our findings in a mouse COVID-19 model, confirmed virome differences and bacteriome dysbiosis due to SARS-CoV-2 infection, and observed that immune/infection-related genes were differentially expressed in gut epithelial cells during infection, possibly explaining the virome and bacteriome dynamics. Our results suggest that the components of the microbiome, including the bacteriome and virome, are affected by SARS-CoV-2 infections, while their compositional signatures could reflect or even contribute to disease severity and recovery processes.