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.

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.

A thermosensitive hydrogel formulation of phage and colistin combination for the management of multidrug-resistant Acinetobacter baumannii wound infections.

Chronic skin wounds are often associated with multidrug-resistant bacteria, impeding the healing process. Bacteriophage (phage) therapy has been revitalized as a promising strategy to counter the growing concerns of antibiotic resistance. However, phage monotherapy also faces several application drawbacks, such as a narrow host spectrum, the advent of resistant phenotypes and poor stability of phage preparations. Phage-antibiotic synergistic (PAS) combination therapy has recently been suggested as a possible approach to overcome these shortcomings. In the present study, we employed a model PAS combination containing a vB_AbaM-IME-AB2 phage and colistin to develop stable wound dressings of PAS to mitigate infections associated with Acinetobacter baumannii. A set of thermosensitive hydrogels were synthesized with varying amounts of Pluronic® F-127 (PF-127 at 15, 17.5 and 20 w/w%) modified with/without 3 w/w% hydroxypropyl methylcellulose (HPMC). Most hydrogel formulations had a gelation temperature around skin temperature, suitable for topical application. The solidified gels were capable of releasing the encapsulated phage and colistin in a sustained manner to kill bacteria. The highest bactericidal effect was achieved with the formulation containing 17.5% PF-127 and 3% HPMC (F5), which effectively killed bacteria in both planktonic (by 5.66 log) and biofilm (by 3 log) states and inhibited bacterial regrowth. Good storage stability of F5 was also noted with negligible activity loss after 9 months of storage at 4 °C. The ex vivo antibacterial efficacy of the F5 hydrogel formulation was also investigated in a pork skin wound infection model, where it significantly reduced the bacterial burden by 4.65 log. These positive outcomes warrant its further development as a topical PAS-wound dressing.

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.

Genomic analysis of K47-type Klebsiella pneumoniae phage IME305, a newly isolated member of the genus Teetrevirus.

We isolated a K47-type Klebsiella pneumoniae phage from untreated hospital sewage: vB_KpnP_IME305 (GenBank no. OK149215). Next-generation sequencing (NGS) demonstrated that IME305 has a double-stranded DNA genome of 38,641 bp with 50.9% GC content. According to BLASTn comparisons, the IME305 genome sequence shares similarity with that of Klebsiella phage 6998 (97.37% identity and 95% coverage). IME305 contains 45 open reading frames (ORFs) and no rRNA, tRNA, or virulence-related gene sequences. Bioinformatic analysis showed that IME305 belongs to the phage subfamily Studiervirinae and genus Teetrevirus.

Macrophage-based delivery of anti-fibrotic proteins alleviates bleomycin-induced pulmonary fibrosis in mice.

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease characterized by chronic, progressive, and fibrotic lung injury. Although remarkable progress has been made toward understanding the pathogenesis of PF, finding more effective treatments for this fatal disease remains a challenge. In this study, we describe an innovative macrophage-based approach to deliver anti-fibrotic protein to the lung and inhibit PF in a mouse model of bleomycin (BLM)-induced lung injury. We engineered macrophages to continuously secrete three types of proteins: interleukin-10, which prevents inflammation; TGFRcFc, a soluble truncated TGF-ßR2 that blocks TGF-ß; and CD147, which induces matrix metalloproteinases (MMPs) and causes collagen degradation. Infusing these engineered macrophages into the lungs of BLM-induced PF mouse models in an optimal pattern significantly ameliorated PF in mice. Specifically, the most effective therapeutic outcome was achieved by infusing IL-10-secreting macrophages on day 1, followed by TGFRcFc-secreting macrophages on day 7 and CD147-secreting macrophages on day 14 into the same mice after BLM treatment. Our data suggest that macrophage-based delivery of anti-fibrotic proteins to the lungs is a promising therapy for fibrotic lung disorders.

Bronchoalveolar Lavage Fluid from Chronic Obstructive Pulmonary Disease Patients Increases Neutrophil Chemotaxis Measured by a Microfluidic Platform.

Chronic obstructive pulmonary disease (COPD) is a persistent and progressive respiratory disorder characterized by expiratory airflow limitation caused by chronic inflammation. Evidence has shown that COPD is correlated with neutrophil chemotaxis towards the airways, resulting in neutrophilic airway inflammation. This study aimed to evaluate neutrophil chemotaxis in bronchoalveolar lavage fluid (BALF) from COPD patients using a high-throughput nine-unit microfluidic platform and explore the possible correlations between neutrophil migratory dynamics and COPD development. The results showed that BALF from COPD patients induced stronger neutrophil chemotaxis than the Control BALF. Our results also showed that the chemotactic migration of neutrophils isolated from the blood of COPD patients was not significantly different from neutrophils from healthy controls, and neutrophil migration in three known chemoattractants (fMLP, IL-8, and LTB4) was not affected by glucocorticoid treatment. Moreover, comparison with clinical data showed a trend of a negative relationship between neutrophil migration chemotactic index (C. I.) in COPD BALF and patient's spirometry data, suggesting a potential correlation between neutrophil migration and the severity of COPD. The present study demonstrated the feasibility of using the microfluidic platform to assess neutrophil chemotaxis in COPD pathogenesis, and it may serve as a potential marker for COPD evaluation in the future.

Sequential treatment effects on phage-antibiotic synergistic application against multi-drug-resistant Acinetobacter baumannii.

Bacteriophage (phage) therapy, exploiting phages which are the natural enemies of bacteria, has been re-introduced to treat multidrug-resistant (MDR) bacterial infections. However, some intrinsic drawbacks of phages are overshadowing their clinical use, particularly the narrow host spectrum and rapid emergence of resistance upon treatment. The use of phage-antibiotic combinations exhibiting synergistic bacterial killing [termed 'phage-antibiotic synergy' (PAS)] has therefore been proposed. It is well reported that the types and doses of phages and antibiotics are critical in achieving PAS. However, the impact of treatment order has received less research attention. As such, this study used an Acinetobacter baumannii phage vB_AbaM-IME-AB2 and colistin as a model PAS combination to elucidate the order effects in-vitro. While application of the phage 8 h before colistin treatment demonstrated the greatest antibacterial synergy, it failed to prevent the development of phage resistance. On the other hand, simultaneous application and antibiotic followed by phage application were able to suppress/delay the development of resistance effectively, and simultaneous application demonstrated superior antibacterial and antibiofilm activities. Further in-vivo investigation is required to confirm the impact of treatment order on PAS.

Longitudinal Dynamics of Cellular Responses in Recovered COVID-19 Patients.

Safe and effective vaccines and therapeutics based on the understanding of antiviral immunity are urgently needed to end the COVID-19 pandemic. However, the understanding of these immune responses, especially cellular immune responses to SARS-CoV-2 infection, is limited. Here, we conducted a cohort study of COVID-19 patients who were followed and had blood collected to characterize the longitudinal dynamics of their cellular immune responses. Compared with healthy controls, the percentage of activation of SARS-CoV-2 S/N-specific T cells in recovered patients was significantly higher. And the activation percentage of S/N-specific CD8+ T cells in recovered patients was significantly higher than that of CD4+ T cells. Notably, SARS-CoV-2 specific T-cell responses were strongly biased toward the expression of Th1 cytokines, included the cytokines IFNγ, TNFα and IL2. Moreover, the secreted IFNγ and IL2 level in severe patients was higher than that in mild patients. Additionally, the number of IFNγ-secreting S-specific T cells in recovered patients were higher than that of N-specific T cells. Overall, the SARS-CoV-2 S/N-specific T-cell responses in recovered patients were strong, and virus-specific immunity was present until 14-16 weeks after symptom onset. Our work provides a basis for understanding the immune responses and pathogenesis of COVID-19. It also has implications for vaccine development and optimization and speeding up the licensing of the next generation of COVID-19 vaccines.

Characterization and Genomic Analysis of Bacteriophage vB_KpnM_IME346 Targeting Clinical Klebsiella pneumoniae Strain of the K63 Capsular Type.

A Klebsiella pneumoniae bacteriophage (vB_KpnM_IME346) was isolated from a hospital sewage sample. This bacteriophage specifically infects a clinical K. pneumoniae strain with a K63 capsular polysaccharide structure. The phage genome was evaluated by next-generation sequencing, which revealed a linear double-stranded DNA genome consisting of 49,482 base pairs with a G+C content of 49.1%. The latent period of vB_KpnM_IME346 was shown to be 20 min, and the burst size was 25-30 pfu (plaque-forming units)/infected cell. Transmission electron microscopy and phylogenetic analysis showed that the JD001-like phage belongs to the genus Jedunavirus of the family Myoviridae. The newly isolated vB_KpnM_IME346 shows infectivity in the clinical host K. pneumoniae KP576 strain, indicating that it is a promising alternative to antibacterial agents for removing K. pneumoniae from patients.

Landscapes and dynamic diversifications of B-cell receptor repertoires in COVID-19 patients.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the pandemic of coronavirus disease 2019 (COVID-19). Great international efforts have been put into the development of prophylactic vaccines and neutralizing antibodies. However, the knowledge about the B cell immune response induced by the SARS-CoV-2 virus is still limited. Here, we report a comprehensive characterization of the dynamics of immunoglobin heavy chain (IGH) repertoire in COVID-19 patients. By using next-generation sequencing technology, we examined the temporal changes in the landscape of the patient's immunological status and found dramatic changes in the IGH within the patient's immune system after the onset of COVID-19 symptoms. Although different patients have distinct immune responses to SARS-CoV-2 infection, by employing clonotype overlap, lineage expansion, and clonotype network analyses, we observed a higher clonotype overlap and substantial lineage expansion of B cell clones 2-3 weeks after the onset of illness, which is of great importance to B-cell immune responses. Meanwhile, for preferences of V gene usage during SARS-CoV-2 infection, IGHV3-74 and IGHV4-34, and IGHV4-39 in COVID-19 patients were more abundant than those of healthy controls. Overall, we present an immunological resource for SARS-CoV-2 that could promote both therapeutic development as well as mechanistic research.

Metagenomic diagnosis of severe psittacosis using multiple sequencing platforms.

BACKGROUND: Chlamydia psittaci is an avian pathogen that can cause lethal human infections. Diagnosis of C. psittaci pneumonia is often delayed due to nonspecific clinical presentations and limited laboratory diagnostic techniques. RESULTS: The MinION platform established the diagnosis in the shortest time, while BGISEQ-500 generated additional in-depth sequence data that included the rapid characterization of antibiotic susceptibility. Cytopathy appeared only in cell cultures of BALF. BALF yielded a higher bacterial load than sputum or blood, and may be the most suitable clinical specimen for the genomic diagnosis of severe pneumonia. CONCLUSIONS: This study indicated that the benefits of metagenomic sequencing include rapid etiologic diagnosis of unknown infections and the provision of additional relevant information regarding antibiotic susceptibility. The continued optimization and standardization of sampling and metagenomic analysis promise to enhance the clinical utility of genomic diagnosis.

Clinical Analysis of Severe Radiation Pneumonia.

OBJECTIVE: The present study aims to discuss the clinical characteristics, factors, and treatment methods affecting the prognosis in patients with severe radiation pneumonia (RP). METHODS: The radiotherapy status, clinical features, imaging characteristics, laboratory examination results, treatment methods, and prognoses of 34 patients with severe RP treated in our department between January 2011 and July 2017 were retrospectively analyzed. The severe RP grading was based on the Common Terminology Criteria for Adverse Events version 4.0; patients who scored Grade ≥3 were considered to have a severe case of RP. RESULTS: The results of the present study showed that 22 patients had lung cancer, 6 had esophageal cancer, 5 had breast cancer, and 1 had colon cancer with lung metastasis. The total radiation dose was 37.5-66 Gy, and the overall average dose was 53 Gy; the average dose in the patients who died was 52.9 Gy. A total of 28 patients presented with a cough and sputum as the initial symptom, and 24 presented with wheezing as an accompanying symptom; of the 24 patients, 8 experienced fever, 2 experienced wheezing as the only symptom, 1 had chest pain, and 1 had chest tightness. In 26 patients, the changes were in the radiation field, and in 8 cases, the changes appeared both inside and outside the radiation field. After the use of glucocorticoid methylprednisolone, respiratory support, and anti-infection treatment, 18 patients were cured, 8 showed a condition improvement, and 8 died. CONCLUSION: The prognosis of severe RP was not significantly correlated with the administered radiation dose; however, lung cancer, a high Acute Physiology and Chronic Health Evaluation score, and delayed diagnosis were risk factors for patient death. However, a combination of antibiotic therapy, ventilator-assisted respiration, and steroid therapy could improve patient prognosis.

Development of thermosensitive hydrogel wound dressing containing Acinetobacter baumannii phage against wound infections.

With the emergence of multidrug resistance (MDR) bacteria, wound infection continues to be a challenging problem and represents a considerable healthcare burden. This study aims to evaluate the applicability of a phage loaded thermosensitive hydrogel in managing wound infections caused by MDR Acinetobacter baumannii, using IME-AB2 phage and MDR-AB2 as the model phage and bacteria, respectively. Excellent storage stability of the IME-AB2 phage in a ~18 wt% Poloxamer 407 (P407) hydrogel solution was first demonstrated with negligible titer loss (~0.5 log) in 24 months at 4 °C. The incorporated phage was released in a sustained manner with a cumulative release of 60% in the first 24 h. The in vitro bacterial killing efficiency of phage gel and phage suspension at 37 °C demonstrated >5 log10 CFU/ml reduction against A. baumannii. A comparable biofilm elimination capacity was also noted between the phage gel and phage suspension (59% and 45% respectively). These results suggested that the incorporation of phage into the hydrogel not only had insignificant impacts on the bacterial killing efficiency of phage, but also act as a phage depot to maintain higher phage titer at the infectious site for a prolong period for more effective treatment. We also found that the hydrogel formulation significantly suppressed microbial survival in an ex vivo wound infection model using pig skin (90% reduction in bacterial counts was achieved after 4 h treatment). In summary, our results demonstrated that the P407-based phage-loaded thermosensitive hydrogel is a simple and promising phage formulation for the management of wound infections.

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.

Phase 2a, open-label, dose-escalating, multi-center pharmacokinetic study of favipiravir (T-705) in combination with oseltamivir in patients with severe influenza.

BACKGROUND: The pharmacokinetics and appropriate dose regimens of favipiravir are unknown in hospitalized influenza patients; such data are also needed to determine dosage selection for favipiravir trials in COVID-19. METHODS: In this dose-escalating study, favipiravir pharmacokinetics and tolerability were assessed in critically ill influenza patients. Participants received one of two dosing regimens; Japan licensed dose (1600 mg BID on day 1 and 600 mg BID on the following days) and the higher dose (1800 mg/800 mg BID) trialed in uncomplicated influenza. The primary pharmacokinetic endpoint was the proportion of patients with a minimum observed plasma trough concentration (Ctrough) ≥20 mg/L at all measured time points after the second dose. RESULTS: Sixteen patients were enrolled into the low dose group and 19 patients into the high dose group of the study. Favipiravir Ctrough decreased significantly over time in both groups (p <0.01). Relative to day 2 (48 hrs), concentrations were 91.7% and 90.3% lower in the 1600/600 mg group and 79.3% and 89.5% lower in the 1800/800 mg group at day 7 and 10, respectively. In contrast, oseltamivir concentrations did not change significantly over time. A 2-compartment disposition model with first-order absorption and elimination described the observed favipiravir concentration-time data well. Modeling demonstrated that less than 50% of patients achieved Ctrough ≥20 mg/L for >80% of the duration of treatment of the two dose regimens evaluated (18.8% and 42.1% of patients for low and high dose regimen, respectively). Increasing the favipravir dosage predicted a higher proportion of patients reaching this threshold of 20 mg/L, suggesting that dosing regimens of ≥3600/2600 mg might be required for adequate concentrations. The two dosing regimens were well-tolerated in critical ill patients with influenza. CONCLUSION: The two dosing regimens proposed for uncomplicated influenza did not achieve our pre-defined treatment threshold.