Construction of viral-based expression vectors for high-level production of human interferon alpha 2b in plants.

Human interferon (hINF) alpha 2b is clinically important pharmaceutical product included in combinatory therapy against chronic hepatitis C and B and complex therapy against several cancer diseases. Here, we created the genetic constructions, based on genome elements of potato virus X (PVX), carrying the infα2b gene for transient expression in plant cells. The created plasmid vector constructions were tested through Agrobacterium-mediated transient gene expression method in two plant species-Nicotiana benthamiana and Ocimum basilicum (sweet basil). Production of recombinant hINF alpha 2b was more efficient in N. benthamiana than that in O. basilicum plants. The average yield of hINF alpha 2b produced in N. benthamiana plants was 0.56 mg/g of fresh leaf weight (FW) or 6% of the total soluble cell proteins (TSP). The maximal level reached up to 1.2 mg/g FW or 9% TSP. We estimated that about 0.67 mg of hINF can be obtained from one N. benthamiana plant. The yield of hINF alpha 2b obtained with the PVX-based expression cassette was about 80 times higher than the yield of hINF alpha 2b obtained with a simple expression cassette in which the infα2b gene was controlled by the 35S promoter of cauliflower mosaic virus. KEY POINTS: • PVX-based expression vectors provide efficient transient expression of infα2b gene • N. benthamiana plants can produce human interferon alpha 2b at high levels • The yield of the hINF α2b reached up to 1.2 mg/g of fresh leaf weight.

Chicken Interferon-Alpha and -Lambda Exhibit Antiviral Effects against Fowl Adenovirus Serotype 4 in Leghorn Male Hepatocellular Cells.

Hydropericardium hepatitis syndrome (HHS) is primarily caused by fowl adenovirus serotype 4 (FAdV-4), causing high mortality in chickens. Although vaccination strategies against FAdV-4 have been adopted, HHS still occurs sporadically. Furthermore, no effective drugs are available for controlling FAdV-4 infection. However, type I and III interferon (IFN) are crucial therapeutic agents against viral infection. The following experiments were conducted to investigate the inhibitory effect of chicken IFN against FadV-4. We expressed recombinant chicken type I IFN-α (ChIFN-α) and type III IFN-λ (ChIFN-λ) in Escherichia coli and systemically investigated their antiviral activity against FAdV-4 infection in Leghorn male hepatocellular (LMH) cells. ChIFN-α and ChIFN-λ dose dependently inhibited FAdV-4 replication in LMH cells. Compared with ChIFN-λ, ChIFN-α more significantly inhibited viral genome transcription but less significantly suppressed FAdV-4 release. ChIFN-α- and ChIFN-λ-induced IFN-stimulated gene (ISG) expression, such as PKR, ZAP, IRF7, MX1, Viperin, IFIT5, OASL, and IFI6, in LMH cells; however, ChIFN-α induced a stronger expression level than ChIFN-λ. Thus, our data revealed that ChIFN-α and ChIFN-λ might trigger different ISG expression levels, inhibiting FAdV-4 replication via different steps of the FAdV-4 lifecycle, which furthers the potential applications of IFN antiviral drugs in chickens.

Interferon-α receptor antisense oligonucleotides reduce neuroinflammation and neuropathology in a mouse model of cerebral interferonopathy.

Chronic and elevated levels of the antiviral cytokine IFN-α in the brain are neurotoxic. This is best observed in patients with genetic cerebral interferonopathies such as Aicardi-Goutières syndrome. Cerebral interferonopathies typically manifest in early childhood and lead to debilitating disease and premature death. There is no cure for these diseases with existing treatments largely aimed at managing symptoms. Thus, an effective therapeutic strategy is urgently needed. Here, we investigated the effect of antisense oligonucleotides targeting the murine IFN-α receptor (Ifnar1 ASOs) in a transgenic mouse model of cerebral interferonopathy. Intracerebroventricular injection of Ifnar1 ASOs into transgenic mice with brain-targeted chronic IFN-α production resulted in a blunted cerebral interferon signature, reduced neuroinflammation, restoration of blood-brain barrier integrity, absence of tissue destruction, and lessened neuronal damage. Remarkably, Ifnar1 ASO treatment was also effective when given after the onset of neuropathological changes, as it reversed such disease-related features. We conclude that ASOs targeting the IFN-α receptor halt and reverse progression of IFN-α-mediated neuroinflammation and neurotoxicity, opening what we believe to be a new and promising approach for the treatment of patients with cerebral interferonopathies.

Detection of interferon alpha and beta receptor subunit 1 (IFNAR1) loss-of-function Glu386∗ variant by tri-allelic genotyping.

Mutations of the human interferon alpha and beta receptor subunit 1 (IFNAR1) gene are associated with severe viral infections. Individuals homozygous for the Glu386∗ variant have impaired type I interferon signalling and can suffer severe illness when exposed to certain viruses and live attenuated virus vaccines. Glu386∗ heterozygotes are clinically unaffected, but can pass the variant allele to their descendants. We aimed to develop an assay that can identify IFNAR1 Glu386∗ homozygotes and heterozygotes to support urgent clinical diagnosis, and that can use dried blood spots (DBS) sent at ambient temperature to overcome geographical logistical challenges in the South Pacific region. The tri-allelic genotyping assay interrogates a single nucleotide polymorphism (rs201609461) located in IFNAR1. The reference allele G encodes for wild-type IFNAR1. Minor alleles A (c.1156G>A) and T (c.1156G>T) encode for Glu386Lys and a truncated IFNAR1 protein (p.Glu386∗), respectively. Synthetic oligonucleotides were mixed in equal molar ratio to create six different genotypes which were randomly assigned to 960 genotyping reactions by R software. Three different fluorescence probes were designed to discriminate the three alleles (G, T and A) within a pair of flanking primers in a single genotyping reaction. The assay discriminated all three alleles using DBS from Guthrie cards randomly spiked with synthetic oligonucleotides. We correctly identified all the different genotypes in 960 reactions in these blinded experiments. These findings validate the genotyping assay for rapidly identifying the IFNAR1 Glu386∗ variant from DBS.

Engineering a New IFN-ApoA-I Fusion Protein with Low Toxicity and Prolonged Action.

Recombinant human interferon alpha-2b (rIFN) is widely used in antiviral and anticancer immunotherapy. However, the high efficiency of interferon therapy is accompanied by a number of side effects; this problem requires the design of a new class of interferon molecules with reduced cytotoxicity. In this work, IFN was modified via genetic engineering methods by merging it with the blood plasma protein apolipoprotein A-I in order to reduce acute toxicity and improve the pharmacokinetics of IFN. The chimeric protein was obtained via biosynthesis in the yeast P. pastoris. The yield of ryIFN-ApoA-I protein when cultivated on a shaker in flasks was 30 mg/L; protein purification was carried out using reverse-phase chromatography to a purity of 95-97%. The chimeric protein demonstrated complete preservation of the biological activity of IFN in the model of vesicular stomatitis virus and SARS-CoV-2. In addition, the chimeric form had reduced cytotoxicity towards Vero cells and increased cell viability under viral load conditions compared with commercial IFN-a2b preparations. Analysis of the pharmacokinetic profile of ryIFN-ApoA-I after a single subcutaneous injection in mice showed a 1.8-fold increased half-life of the chimeric protein compared with ryIFN.

Fatal COVID-19 Infection in Two Children with STAT1 Gain-of-Function.

While SARS-CoV-2 infection causes a mild disease in most children, SARS-CoV-2 infection may be lethal in a few of them. In the defense against SARS-CoV-2, type I interferons are key players, and several studies have identified a defective or neutralized interferon response as the cause of overwhelming viral infection. However, inappropriate, untimely, or excessive interferon production may also be detrimental to the host. Here, we describe two patients with STAT1 gain-of-function (GOF), a known type I interferonopathy, who died of COVID-19. Whole-exome sequencing and interferon-gamma-activated sequence (GAS) and interferon-sensitive responsive element (ISRE) reporter assay were performed to identify and characterize STAT1 variants. Patient 1 developed hemophagocytic lymphohistiocytosis (HLH) in the context of COVID-19 infection and died in less than a week at the age of 4 years. Patient 2 developed a high fever, cough, and hypoxemia and succumbed to COVID-19 pneumonia at the age of 5 years. Two heterozygous missense variants, p.E563Q and p.K344E, in STAT1 were identified. Functional validation by reporter assay and immunoblot confirmed that both variants are gain-of-function (GOF). GOF variants transiently expressing cells exhibited enhanced upregulation of downstream genes, including ISG15, MX1, and OAS1, in response to IFN-α stimulation. A catastrophic course with HLH or acute respiratory failure is thought to be associated with inappropriate immunoregulatory mechanisms to handle SARS-CoV-2 in STAT1 GOF. While most patients with inborn errors of immunity who developed COVID-19 seem to handle it well, these cases suggest that patients with STAT1-GOF might be at risk of developing fatal complications due to SARS-CoV-2.

A novel TRIM22 gene polymorphism promotes the response to PegIFNα therapy through cytokine-cytokine receptor interaction signaling pathway in chronic hepatitis B.

IMPORTANCE: Pegylated interferon alfa (PegIFNα) has limited efficacy in the treatment of chronic hepatitis B (CHB). Although many biomarkers related to hepatitis B virus (HBV) have been proposed to stratify patients, the response rate to PegIFNα is still unsatisfactory. Herein, our data suggest that the single-nucleotide polymorphism (SNP) rs10838543 in TRIM22 potentiates a positive clinical response to PegIFNα treatment in patients with hepatitis B e antigen-positive CHB by increasing the levels of IFNL1, CCL3, and CCL5. These observations can help guide treatment decisions for patients with CHB to improve the response rate to PegIFNα.

Management of classical Philadelphia chromosome-negative myeloproliferative neoplasms in Asia: consensus of the Asian Myeloid Working Group.

Myeloproliferative neoplasms (MPN) are a heterogeneous group of clonal hematopoietic stem cell disorders characterized clinically by the proliferation of one or more hematopoietic lineage(s). The classical Philadelphia-chromosome (Ph)-negative MPNs include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The Asian Myeloid Working Group (AMWG) comprises representatives from fifteen Asian centers experienced in the management of MPN. This consensus from the AMWG aims to review the current evidence in the risk stratification and treatment of Ph-negative MPN, to identify management gaps for future improvement, and to offer pragmatic approaches for treatment commensurate with different levels of resources, drug availabilities and reimbursement policies in its constituent regions. The management of MPN should be patient-specific and based on accurate diagnostic and prognostic tools. In patients with PV, ET and early/prefibrotic PMF, symptoms and risk stratification will guide the need for early cytoreduction. In younger patients requiring cytoreduction and in those experiencing resistance or intolerance to hydroxyurea, recombinant interferon-α preparations (pegylated interferon-α 2A or ropeginterferon-α 2b) should be considered. In myelofibrosis, continuous risk assessment and symptom burden assessment are essential in guiding treatment selection. Allogeneic hematopoietic stem cell transplantation (allo-HSCT) in MF should always be based on accurate risk stratification for disease-risk and post-HSCT outcome. Management of classical Ph-negative MPN entails accurate diagnosis, cytogenetic and molecular evaluation, risk stratification, and treatment strategies that are outcome-oriented (curative, disease modification, improvement of quality-of-life).

Interferon gene therapy with nadofaragene firadenovec for bladder cancer: from bench to approval.

Bladder cancer is a prevalent malignancy with limited therapeutic options, particularly for patients who are unresponsive to Bacillus Calmette-Guérin (BCG). The approval of interferon-α (IFNα) gene therapy with nadofaragene firadenovec (Adstiladrin®), the first gene therapy for genitourinary malignancies, has provided a promising alternative. This article reviews the research and milestones that led to the development and approval of nadofaragene firadenovec. Bladder cancer is well-suited for gene therapy due to direct access to the bladder and the availability of urine and tissue samples for monitoring. Early challenges included effective gene transfer across the urothelium, which was overcome initially by modulating the expression of coxsackie/adenovirus receptor (CAR) and, ultimately, by disrupting the urothelial barrier with Syn3. Nadofaragene firadenovec is a modified adenoviral vector carrying the IFNα gene. Clinical trials have shown promising results, with high response rates and manageable adverse events. Ongoing research focuses on improving patient selection, identifying biomarkers for response prediction, exploring alternative vectors for enhanced transfection efficiency, and developing combination strategies targeting resistance mechanisms. The approval of nadofaragene firadenovec marks a significant milestone in the field of gene therapy for bladder cancer, and future developments hold promise for further enhancing its efficacy and impact.

Upregulated SPAG6 correlates with increased STAT1 and is associated with reduced sensitivity of interferon-α response in BCR::ABL1 negative myeloproliferative neoplasms.

Sperm-associated antigen 6 (SPAG6) has been identified as an oncogene or tumor suppressor in various types of human cancer. However, the role of SPAG6 in BCR::ABL1 negative myeloproliferative neoplasms (MPNs) remains unclear. Herein, we found that SPAG6 was upregulated at the mRNA level in primary MPN cells and MPN-derived leukemia cell lines. The SPAG6 protein was primarily located in the cytoplasm around the nucleus and positively correlated with ß-tubulin expression. In vitro, forced expression of SPAG6 increased cell clone formation and promoted G1 to S cell cycle progression. Downregulation of SPAG6 promoted apoptosis, reduced G1 to S phase transition, and impaired cell proliferation and cytokine release accompanied by downregulated signal transducer and activator of transcription 1 (STAT1) expression. Furthermore, the inhibitory effect of interferon-α (INF-α) on the primary MPN cells with high SPAG6 expression was decreased. Downregulation of SPAG6 enhanced STAT1 induction, thus enhancing the proapoptotic and cell cycle arrest effects of INF-α both in vitro and in vivo. Finally, a decrease in SPAG6 protein expression was noted when the STAT1 signaling was blocked. Chromatin immunoprecipitation assays indicated that STAT1 protein could bind to the SPAG6 promoter, while the dual-luciferase reporter assay indicated that STAT1 could promote the expression of SPAG6. Our results substantiate the relationship between upregulated SPAG6, increased STAT1, and reduced sensitivity to INF-α response in MPN.

Expression and purification of human interferon alpha 2a (IFNα2a) in the methylotrophic yeast Pichia pastoris.

Human interferon alpha 2a (IFNα2a) is a secreted glycoprotein that exerts a wide spectrum of biological effects, such as triggering of antiviral, antitumor and immunosuppressive responses. IFNα2a is used as pharmaceutical polypeptide in chronic hepatitis C virus (HCV) infection, chronic myelogenous leukemia, advanced renal cell carcinoma, and metastatic malignant melanoma. So far, the pharmaceutical polypeptide of this cytokine is produced in prokaryotic expression systems (E. coli). Here we report the expression and purification of recombinant human IFNα2a in the methylotrophic yeast Pichia pastoris. The cDNA encoding for human IFNα2a, modified to bear the P. pastoris codon bias, was cloned into the pPinkα-HC vector in order to be expressed as a secreted protein upon induction. Proper expression and secretion of recombinant human IFNα2a (approximately 19 kDa) was confirmed by PCR-sequencing, SDS-PAGE and Western blot analysis following methanol-induced expression in a number of individual transformed strains. Purification of the recombinant protein was performed by affinity chromatography, achieving a robust yield of purified active form. The purified recombinant protein showed an impressive stability to thermal denaturation as observed by Differential Scanning Fluorimetry. The biological activity of the P. pastoris-produced IFNα2a was confirmed in A549 and HT29 cells by monitoring transcriptional up-regulation of a panel of known interferon-stimulated genes (ISGs). Our results document that the P. pastoris expression system is a suitable system for producing biologically functional IFNα2a in a secreted form.

Aicardi-Goutières syndrome: A monogenic type I interferonopathy.

Aicardi-Goutières syndrome (AGS) is a rare monogenic autoimmune disease that primarily affects the brains of children patients. Its main clinical features include encephalatrophy, basal ganglia calcification, leukoencephalopathy, lymphocytosis and increased interferon-α (IFN-α) levels in the patient's cerebrospinal fluid (CSF) and serum. AGS may be caused by mutations in any one of nine genes (TREX1, RNASEH2A, RNASEH2B, RNASEH2C, SAMHD1, ADAR1, IFIH1, LSM11 and RNU7-1) that result in accumulation of self-nucleic acids in the cytoplasm or aberrant sensing of self-nucleic acids. This triggers overproduction of type I interferons (IFNs) and subsequently causes AGS, the prototype of type I interferonopathies. This review describes the discovery history of AGS with various genotypes and provides the latest knowledge of clinical manifestations and causative genes of AGS. The relationship between AGS and type I interferonopathy and potential therapeutic methods for AGS are also discussed in this review.

A Transcriptome Array-Based Approach Links Proteinuria and Distinct Molecular Signatures to Intrarenal Expression of Type I Interferon IFNA5 in Lupus Nephritis.

In systemic lupus erythematosus (SLE), the relevance of non-hematopoietic sources of type I interferon in human autoimmunity has recently been recognized. Particularly, type I interferon production precedes autoimmunity in early skin lesions related to SLE. However, the relevance of intrarenal type I interferon expression has not been shown in lupus nephritis. From transcriptome array datasets, median-centered log2 mRNA expression levels of IFNα (IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21), IFNω (IFNW1), and IFNß (IFNB1) in lupus nephritis were extracted specifically from microdissected tubulointerstitial (n = 32) and glomerular compartments (n = 32). We found an association between proteinuria and tubulointerstitial expression of type I interferon IFNA5 (p = 0.0142), while all others were not significantly associated. By contrast, no such correlation was observed between proteinuria and any type I interferon expression in the glomerular compartment in lupus nephritis. Interestingly, there was no difference between female and male patients (p = 0.8237) and no association between type I interferon IFNA5 expression and kidney function or lupus nephritis progression. Finally, we identified distinct molecular signatures involved in transcriptional regulation (GLI protein-regulated transcription, IRF7 activation, and HSF1-dependent transactivation) and receptor signaling (BMP signaling and GPCR ligand binding) in association with tubulointerstitial expression of type I interferon IFNA5 in the kidney. In summary, this transcriptome array-based approach links proteinuria to the tubulointerstitial expression of type I interferon IFNA5 in lupus nephritis. Because type I interferon receptor subunit I antagonism has recently been investigated in active SLE, the current study further emphasizes the role of type I interferons in lupus nephritis and might also be of relevance for mechanistic studies.

Effect of duck interferon-α and an anti-cap protein polyclonal antibody against duck circovirus.

Duck circovirus (DuCV) is one of the most prevalent viruses in the duck breeding industry, and causes persistent infection and severe immunosuppression. Currently, there is a serious lack of prevention and control measures and no commercial vaccine against DuCV. Therefore, effective antiviral drugs are important for treating DuCV infection. Interferon (IFN) is an important component of antiviral innate immunity, but it remains unclear whether duck IFN-α has a clinical effect against DuCV. Antibody therapy is an important way to treat viral infections. The DuCV structural protein (cap) is immunogenic, and it remains to be determined whether an anti-cap protein antibody can effectively block DuCV infection. In this study, the duck IFN-α gene and the DuCV structural protein cap gene were cloned, expressed and purified in Escherichia coli to prepare duck recombinant IFN-α and the cap protein. Then, rabbits were immunized with the recombinant cap protein to prepare a rabbit polyclonal antibody. This study investigated the antiviral effect of duck recombinant IFN-α and the anti-cap protein antibody and their combined effect on Cherry Valley ducks infected with DuCV. The results showed that the treatment significantly alleviated the clinical symptoms of immune organ atrophy and immunosuppression compared with the control. The histopathological damage of the target organs was alleviated, and replication of DuCV in the immune organs was significantly inhibited. The treatment also reduced the damage caused by DuCV to the liver and immune function, and increased the level of the DuCV antibody in the blood, thereby improving antiviral activity. Notably, the combination of duck IFN-α and the polyclonal antibody completely blocked DuCV infection after 13 days under the experimental conditions, showing a better inhibitory effect on DuCV infection than single treatments. These results showed that duck recombinant IFN-α and the anti-cap protein antibody can be used as antiviral drugs to clinically treat and control DuCV infection, particularly the vertical transmission of the virus in breeding ducks.

ChIFNα regulates adventitious root development in Lotus japonicus via an auxin-mediated pathway.

Adventitious roots (ARs), developing from non-root tissue, play an important role in some plants. Here, the molecular mechanism of AR differentiation in Lotus japonicus L. (L. japonicus) with the transformed chicken interferon alpha gene (ChIFNα) encoding cytokine was studied. ChIFNα transgenic plants (TP) were identified by GUS staining, PCR, RT-PCR, and ELISA. Up to 0.175 µg/kg rChIFNα was detected in TP2 lines. Expressing rChIFNα promotes AR development by producing longer roots than controls. We found that the effect was enhanced with the auxin precursor IBA treatment in TP. IAA contents, POD, and PPO activities associated with auxin regulation were higher than wild type (WT) in TP and exogenous ChIFNα treatment plants. Transcriptome analysis revealed 48 auxin-related differentially expressed genes (DEGs) (FDR < 0.05), which expression levels were verified by RT-qPCR analysis. GO enrichment analysis of DEGs also highlighted the auxin pathway. Further analysis found that ChIFNα significantly enhanced auxin synthesis and signaling mainly with up-regulated genes of ALDH, and GH3. Our study reveals that ChIFNα can promote plant AR development by mediating auxin regulation. The findings help explore the role of ChIFNα cytokines and expand animal gene sources for the molecular breeding of growth regulation of forage plants.

Biological activity of recombinant bovine IFN-α and inhibitory effect on BVDV in vitro.

Type I interferon has great broad-spectrum antiviral ability and immunomodulatory function, and its receptors are expressed in almost all types of cells. Bovine viral diarrhea virus (BVDV) is an important pathogen causing significant economic losses in cattle. In this study, a recombinant expression plasmid carrying bovine interferon-α(BoIFN-α)gene was constructed and transformed into E. coli BL21 (DE3) competent cells. SDS-PAGE and Westernblotting analysis showed that the recombinant BoIFN-α protein (rBoIFN-α) was successfully expressed. It is about 36KD and exists in the form of inclusion body. When denatured, purified and renatured rBoIFN-α protein stimulated MDBK cells, the expression of interferon stimulating genes (ISGs) such as ISG15, OAS1, IFIT1, Mx1 and IFITM1 were significantly up-regulated, and reached the peak at 12 h (P< 0.001). MDBK cells were infected with BVDV with moi of 0.1 and 1.0, respectively. The virus proliferation was observed after pretreatment with rBoIFN-α protein and post-infection treatment. The results showed that the denatured, purified and renatured BoIFN-α protein had good biological activity and could inhibit the replication of BVDV in MDBK cells in vitro, which provided a basis for BoIFN-α as an antiviral drug, immune enhancer and clinical application of BVDV.

In Vitro Evaluation of Apoptosis, Inflammation, Angiogenesis, and Neuroprotection Gene Expression in Retinal Pigmented Epithelial Cell Treated with Interferon α-2b.

Angiogenesis, retinal neuropathy, and inflammation are the main molecular features of diabetic retinopathy (DR) and should be taken into consideration for potential treatment approaches. Retinal pigmented epithelial (RPE) cells play a major role in DR progression. This study evaluated the in vitro effect of interferon (IFN) α-2b on the expression of genes involved in apoptosis, inflammation, neuroprotection, and angiogenesis in RPE cells. RPE cells were cocultured with IFN α-2b at 2 doses (500 and 1,000 IU) and treatment periods (24 and 48 h). The quantitative relative expression of genes (BCL-2, BAX, BDNF, VEGF, and IL-1b) was evaluated in the treated versus control cells through real-time polymerase chain reaction (PCR). The result of this study demonstrated that IFN treatment at 1,000 IU (48 h) led to significant upregulation of BCL-2, BAX, BDNF, and IL-1b; however, the BCL-2/BAX ratio was not statistically altered from 1:1, in any of the treatment patterns. We also showed that VEGF expression was downregulated in RPE cells treated with 500 IU for 24 h. It can be concluded that IFN α-2b was safe (BCL-2/BAX ∼1:1) and enhanced neuroprotection at 1,000 IU (48 h); however-at the same time-IFN α-2b induced inflammation in RPE cells. Moreover, the antiangiogenic effect of IFN α-2b was solely observed in RPE cells treated with 500 IU (24 h). It seems that IFN α-2b in lower doses and short duration exerts antiangiogenic effects and in higher doses and longer duration has neuroprotective and inflammatory effects. Hence, appropriate concentration and duration of treatment, according to the type and stage of the disease, should be considered to achieve success in IFN therapy.

Two Novel Adenovirus Vectors Mediated Differential Antibody Responses via Interferon-α and Natural Killer Cells.

Recombinant adenovirus vectors have been widely used in vaccine development. To overcome the preexisting immunity of human adenovirus type 5 (Ad5) in populations, a range of chimpanzee or rare human adenovirus vectors have been generated. However, these novel adenovirus vectors mediate the diverse immune responses in the hosts. In this study, we explored the immune mechanism of differential antibody responses to SARS-CoV-2 S protein in mice immunized by our previously developed two novel simian adenovirus type 23 (Sad23L) and human adenovirus type 49 (Ad49L), and Ad5 vectored COVID-19 vaccines. Sad23L-nCoV-S and Ad5-nCoV-S vaccines induced the low level of interferon-α (IFN-α) and the high level of antigen-specific antibody responses in wild-type and IFN-α/ß receptor defective (IFNAR-/-) C57 mice, while Ad49L-nCoV-S vaccine induced the high IFN-α and low antibody responses in C57 mice but the high antibody response in IFNAR-/- mice. In addition, the high antibody response was detected in natural killer (NK) cells-blocked but the low in follicular helper T (TFH) cells -blocked C57 mice immunized with Ad49L-nCoV-S vaccine. These results showed that Ad49L vectored vaccine stimulated IFN-α secretion to activate NK cells, and then reduced the number of TFH cells, generation center (GC) B cells and plasma cells, and subsequently reduced antigen-specific antibody production. The different novel adenovirus vectors could be selected for vaccine development according to the need for either humoral or cellular or both immune protections against a particular disease. IMPORTANCE Novel adenovirus vectors are an important antigen delivery platform for vaccine development. Understanding the immune diversity between different adenoviral vectors is critical to design the proper vaccine against an aim disease. In this study, we described the immune mechanism of Sad23L and Ad49L vectored vaccines for raising the equally high specific T cell response but the different level of specific antibody responses in mice. We found that Ad49L-vectored vaccine initiated the high IFN-α and activated NK cells to inhibit antibody response via downregulating the number of CD4+ TFH cells leading to the decline of GC B cells and plasma cells.

Time-dependent recruitment of GAF, ISGF3 and IRF1 complexes shapes IFNα and IFNγ-activated transcriptional responses and explains mechanistic and functional overlap.

To understand in detail the transcriptional and functional overlap of IFN-I- and IFN-II-activated responses, we used an integrative RNAseq-ChIPseq approach in Huh7.5 cells and characterized the genome-wide role of pSTAT1, pSTAT2, IRF9 and IRF1 in time-dependent ISG expression. For the first time, our results provide detailed insight in the timely steps of IFNα- and IFNγ-induced transcription, in which pSTAT1- and pSTAT2-containing ISGF3 and GAF-like complexes and IRF1 are recruited to individual or combined ISRE and GAS composite sites in a phosphorylation- and time-dependent manner. Interestingly, composite genes displayed a more heterogeneous expression pattern, as compared to GAS (early) and ISRE genes (late), with the time- and phosphorylation-dependent recruitment of GAF, ISGF3 and IRF1 after IFNα stimulation and GAF and IRF1 after IFNγ. Moreover, functional composite genes shared features of GAS and ISRE genes through transcription factor co-binding to closely located sites, and were able to sustain IFN responsiveness in STAT1-, STAT2-, IRF9-, IRF1- and IRF9/IRF1-mutant Huh7.5 cells compared to Wt cells. Thus, the ISRE + GAS composite site acted as a molecular switch, depending on the timely available components and transcription factor complexes. Consequently, STAT1, STAT2 and IRF9 were identified as functional composite genes that are part of a positive feedback loop controlling long-term IFNα and IFNγ responses. More important, in the absence of any one of the components, the positive feedback regulation of the ISGF3 and GAF components appeared to be preserved. Together, these findings provide further insight in the existence of a novel ISRE + GAS composite-dependent intracellular amplifier circuit prolonging ISG expression and controlling cellular responsiveness to different types of IFNs and subsequent antiviral activity. It also offers an explanation for the existing molecular and functional overlap between IFN-I- and IFN-II-activated ISG expression.

Transcriptomic Analyses of Exercise Training in Alzheimer's Disease Cerebral Cortex.

BACKGROUND: Research reported exercise could reduce Alzheimer's disease (AD) symptoms in human and animals. However, the molecular mechanism of exercise training via transcriptomic analysis was unclear especially in AD in the cortex area. OBJECTIVE: Investigate potential significant pathways in the cortex area that were affected by exercise during AD. METHODS: RNA-seq analysis, differential expressed genes, functional enrichment analysis, and GSOAP clustering analysis were performed in the isolated cerebral cortex from eight 3xTg AD mice (12 weeks old) randomly and equally divided into control (AD) and exercise training (AD-EX) group. Swimming exercise training in AD-EX group was conducted 30 min/day for 1 month. RESULTS: There were 412 genes significant differentially expressed in AD-EX group compared to AD group. Top 10 upregulated genes in AD-EX group against AD group mostly correlated with neuroinflammation, while top 10 downregulated genes mostly had connection with vascularization, membrane transport, learning memory, and chemokine signal. Pathway analysis revealed the upregulated interferon alpha beta signaling in AD-EX had association with cytokines delivery in microglia cells compared to AD and top 10 upregulated genes involved in interferon alpha beta were Usp18, Isg15, Mx1, Mx2, Stat1, Oas1a, and Irf9; The downregulated extracellular matrix organization in AD-EX had correlation with Aß and neuron cells interaction and Vtn was one of the top 10 downregulated genes involved in this pathway. CONCLUSION: Exercise training influenced 3xTg mice cortex through interferon alpha beta signaling upregulation and extracellular matrix organization downregulation based on transcriptomics analysis.