Human Interferon Research Articles

Effects of Tenofovir Combined with Recombinant Human Interferon α-2b on Negative Conversion Rate, Liver Function, Immune Status, and Drug Safety in Patients with Chronic Hepatitis B: A Systematic Review and Meta-Analysis.

Objective To systematically evaluate the clinical value of tenofovir combined with recombinant human interferon α-2b in the treatment of chronic hepatitis B and to provide evidence-based medicine for its popularization and use. Methods The randomized controlled trials (RCTs) of tenofovir combined with recombinant human interferon α-2b in the online database of PubMed, EMBASE, ScienceDirect, Cochrane Library, China knowledge Network (CNKI), China VIP database, Wanfang database, and China Biomedical Literature Database (CBM) were searched. The data included in this study were extracted by two independent researchers. After extracting the data of the study, the Cochrane manual 5.1.0 standard was used to evaluate the bias risk of all the literature included in this study. RevMan5.4 statistical software was used to analyze the collected data by meta. Results Entecavir combined with recombinant human interferon α-2b can inhibit the activity of HBV polymerase and improve the inflammatory response of the liver. Recombinant human interferon α-2b can regulate immune function by inducing T cell differentiation and maturation and enhancing the production of cytokines. The systematic evaluation showed that entecavir combined with recombinant human interferon α-2b had higher serum HBeAg negative conversion rate, higher drug safety compared with entecavir alone, and improved liver function and immune status. Conclusion Tenofovir combined with recombinant human interferon alpha-2b has a high serum HBeAg negative rate and safety profile for the treatment of chronic hepatitis B. The combination treatment can improve liver function and immune status in patients, but more studies with higher methodological quality and longer duration of intervention are needed for further validation.

Coronaviral Infection and Interferon Response: The Virus-Host Arms Race and COVID-19.

The recent pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in unprecedented morbidity and mortality worldwide. The host cells use a number of pattern recognition receptors (PRRs) for early detection of coronavirus infection, and timely interferon secretion is highly effective against SARS-CoV-2 infection. However, the virus has developed many strategies to delay interferon secretion and disarm cellular defense by intervening in interferon-associated signaling pathways on multiple levels. As a result, some COVID-19 patients suffered dramatic susceptibility to SARS-CoV-2 infection, while another part of the population showed only mild or no symptoms. One hypothesis suggests that functional differences in innate immune integrity could be the key to such variability. This review tries to decipher possible interactions between SARS-CoV-2 proteins and human antiviral interferon sensors. We found that SARS-CoV-2 actively interacts with PRR sensors and antiviral pathways by avoiding interferon suppression, which could result in severe COVID-19 pathogenesis. Finally, we summarize data on available antiviral pharmaceutical options that have shown potential to reduce COVID-19 morbidity and mortality in recent clinical trials.

Clinical Observation of Reduning Combined with Recombinant Human Interferon α-2b in the Treatment of Children with Viral Pneumonia.

Purpose To observe the clinical efficacy of Reduning injection combined with recombinant human interferon α-2b spray in the treatment of children with viral pneumonia. Methods A total of 200 children with viral pneumonia over 2 years old who were admitted to the Pediatrics Department of the Cangzhou Central Hospital from September 2018 to November 2020 were recruited and randomized into the control group and observation group at a ratio of 1 : 1, with 100 cases in each group. The children in the control group were given recombinant human interferon α-2b spray, and the children in the observation group were given Reduning injection on the basis of the control group. The clinical symptoms and signs, clinical efficacy, levels of inflammatory mediators, and drug safety were compared between the two groups. Results The t-test results showed that the disappearance time of body temperature, respiratory rate, pulmonary rales, and cough in the observation group was significantly shorter than that in the control group. The chi-square revealed a significantly higher total effective rate in the observation group vs. the control group. After treatment, the levels of IL-1, IL-6, TNF-α, and CRP in the two groups were lower than the corresponding values before treatment, and greater reduction was observed in the observation group in relative to the control group (both p < 0.05). The two groups have a similar safety profile. Conclusion Reduning combined with recombinant human interferon α-2b produces a remarkable effect in the treatment of children with viral pneumonia, and it ameliorates clinical symptoms and reduces inflammatory response with a good safety profile.

The P2X7 Receptor Antagonist AZ10606120 Does Not Alter Graft-Versus-Host Disease Development and Increases Serum Human Interferon-γ in a Humanized Mouse Model

Allogeneic hematopoietic stem cell transplantation is a curative therapy for hematological malignancies, but its efficacy is limited by graft-versus-host disease (GVHD). This life-threatening disorder develops when donor (graft) immune cells cause inflammatory damage to recipient (host) tissues. The immune cell receptor channel P2X7 and its ligand adenosine 5’-triphosphate (ATP) have been implicated in GVHD pathogenesis. Therefore, this signaling axis represents a potential therapeutic target. This study aimed to investigate if the specific P2X7 antagonist AZ10606120 (AZ10) could prevent GVHD development in a preclinical, humanized mouse model, in which NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice are injected with human peripheral blood mononuclear cells (hPBMCs). Flow cytometric measurements of ATP-induced cation dye uptake revealed that AZ10 blocked P2X7 activity in human RPMI 8226 multiple myeloma cells (IC50 of 1 ± 1 nM) and murine RAW 264.7 macrophages (IC50 of 3 ± 1 nM), as well as primary donor CD4+ and CD8+ T cells. However, AZ10 (2 mg/kg), injected intraperitonealy (i.p.) daily for the first 10 days post-hPBMC injection, did not reduce clinical or histological GVHD development in mice. AZ10 did not impact engraftment of human leukocytes, predominantly CD4+ and CD8+ T cells. However, AZ10 increased serum human interferon gamma (hIFN-γ) concentrations, with CD8+ T cells being the main hIFN-γ producing T cell subset. In conclusion, this study suggests a role for P2X7 activation in impairing hIFN-γ production during GVHD pathogenesis, with the use of P2X7 blockade as a therapeutic strategy warranting further investigation.

Optimization of Voyager V1 (VV1) oncolytic virus systemic delivery in combination with cemiplimab and ipilimumab in patients with melanoma and non–small cell lung cancer (NSCLC).

TPS9595 Background: There is a need for novel immunotherapies to address the patient population that never or no longer responds to immune checkpoint inhibitors (CPI). VV1 is an oncolytic vesicular stomatitis virus engineered to express human interferon beta (IFNβ) to enhance cellular anti-tumor immune responses and tumor selectivity. Phase 1 studies demonstrated VV1 anti-tumor activity in several malignancies with or without a CPI. We are exploring ways to optimize VV1 efficacy in combination with cemiplimab, an anti-PD1 antibody approved for lung, basal and squamous cell skin cancers. Recent clinical data support a 5-fold higher dose of VV1 than was previously explored, and pre-clinical data show synergy between the oncolytic and an anti-CTLA4 antibody, in addition to cemiplimab, supporting exploration of a triplet. What was originally a five-arm study of intravenous (IV) VV1 in combination with IV cemiplimab has been amended to focus on 2 means of optimizing efficacy: use of a higher dose of VV1 and triplet combination in proof-of-concept populations. Methods: We are now enrolling pts with advanced melanoma (after progression on anti-PD1) and plan to include 1st-line NSCLC pts with PD-L1 expression ≥50%. The study is first exploring the preliminary anti-tumor activity, safety, and immunogenic activity of the combination of IV VV1 at a dose of 1.0 x 1011 TCID50 once on D1 followed by IV cemiplimab Q3W starting on D8, or the same regimen with an additional intratumoral injection of VV1 1.0 x 109 TCID50 once on D1 for pts with accessible lesions. Pts receive IV cemiplimab Q3W until confirmed disease progression or intolerable toxicity. Once at least 6 pts have been treated with acceptable safety across the 2 melanoma doublet cohorts using this higher dose of VV1, a 3rd melanoma cohort will open to add a single dose of ipilimumab 50 mg on D1 (all IV triplet). Once 6 melanoma pts have received the triplet safely, the 1st-line NSCLC cohort will open. All cohort decisions are guided by a Data Review Committee. Cohorts will be expanded based on a Simon 2-stage design using a type I error rate of 0.05 and power of 85%. Null ORR is 10% with a target of 35% for 2nd line melanoma and null ORR is 40% in 1st line NSCLC with a target of 70%. Each melanoma cohort will require a response in ≥2 of 10 pts in the 1st stage to add 11 more in the 2nd stage, while NSCLC will first need 5/9 evaluable pts to respond, then an additional 13 to complete the design. Response is assessed at week 7 then Q9W per RECIST v1.1. The study includes serial biopsies in ≥3/10 pts in Stage 1 of each of the IV melanoma cohorts (doublet and triplet therapy), all pts in Stage 2 of these IV melanoma cohorts, and all pts in both Stage 1 and Stage 2 of the IV/IT melanoma cohort, to permit a thorough investigation of the impact of the 3 immunotherapies under investigation. The study is currently ongoing in the USA. Clinical trial information: NCT04291105.

Clinical results of the using leucocyte interferon for the therapy of viral pathology of the oral mucous in somatic patients with combined dentistry pathology

Aim. To conduct a clinical test of the unguent form of human leukocyte interferon in somatic patients with relapsing of herpetic stomatitis.Material and methods. 98 somatic patients with viral pathology of the oral mucous at the age of 20 to 60 years and older were studied to identify the clinical efficiency of the leukocyte interferon. The order of the clinical studies was specified by a special program: preparations were decrypted, decryption was conducted after the analysis of the results of the clinical study. Preparation 1 – placebo (2% emulsion of lidocaine on vinilin), preparation 2 – interferon’s unguent, preparation 3 – 0.5% oxoline unguent. After active implementation of the complex treatment of patients with mucologic pathology were divided into 3 groups: 1st group included 31 patients who got a course of the treatment by preparation 1; 2nd - 34 persons, 2 have taken preparation; 3rd - 33 patients, 3 used preparation.Results. The most expressed clinical effect in the treatment of relapsing of herpetic stomatitis in somatic patients with combined dentistry pathology was shown by preparation 2, which had local anesthetic and anti-inflammation action. Indicators of the recovery of patients who got this preparation in contrast with patients of the third group decreased on average by 2 days, the 1st groups - by more than 1 day.Conclusion. Using oxolin’s unguent and preparation 1 in course of the treatment of herpetic stomatitis did not prevent the development of secondary elements in the oral mucous (24.0% and 44.0% accordingly) in most of the patients. Using preparation 2, new elements on oral mucous were observed only in 6.0% of cases, which shows it good antiviral effect.

Safety and Effectiveness of Inhaling Different Dosage Recombinant Human Interferon α1B for Bronchiolitis in Children: a Systematic Review and Meta-Analysis.

Purpose To systematically evaluate the safety and effectiveness of different dosages of recombinant human interferon α1b (IFNα1b) inhaled for bronchiolitis in children. Methods 7 databases, including PubMed, EMBASE, Cochrane Library, Web of Science, CNKI, Wanfang Database, and VIP, were searched. The search time was from their inception dates to March 28, 2022. A randomized controlled trial (RCT) of 2 μg/kg IFNα1b (low dosage group) monotherapy or in combination with other drugs vs. 4 μg/kg IFNα1b (high dosage group) monotherapy or in combination with the other drugs was included. The risk of bias 2.0 evaluated the RCT's quality, and the grading of recommendations assessment, development and evaluation (GRADE) tool was used for evaluating the overall quality of the evidence. Then, a meta-analysis was performed by RevMan 5.4. Results A total of 13 RCTs with 1719 children were included. The meta-analysis results showed that the high dosage group was significantly shorter than the low dosage group of the duration of hospital stays (MD = −0.40, 95%CI (−0.73, −0.07), P = 0.02) (low quality), three depressions sign disappearing time (MD = −0.60, 95%CI (−1.05, −0.14), P = 0.010) (low quality), and wheeze disappearing time (MD = −0.62, 95%CI (−1.17, −0.06), = 0.03) (low quality). There was no significant difference between the two groups in coughing disappearing time, pulmonary rales disappearing time, wheezing sound disappearing time, or adverse event rates. Conclusions Compared with low dosage IFNα1b, high dosage IFNα1b reduces the duration of hospital stays, the disappearance time of the three depression signs, and the disappearance time of wheeze in the treatment of bronchiolitis in children. Limited by the low quality of the evidence, the conclusions still need to be supported by high-quality studies.

Development and certification of a pharmacopoeial reference standard for primary structure identification of purified recombinant interferon beta-1b by peptide mapping

Medicines based on recombinant human interferons (rhIFNs) beta-1a and beta-1b are used as first-line treatment of multiple sclerosis. Meanwhile, rhIFN beta-1a and beta-1b have structural differences associated with the eukaryotic or prokaryotic expression systems, respectively. Pharmacopoeias require identification of the primary structure of recombinant proteins by peptide mapping, which involves the use of reference material. Currently, there is no international reference standard available for rhIFN beta-1b structural identification. The aim of the study was development and certification of a pharmacopoeial reference standard for identification of the amino acid sequence of purified rhIFN beta-1b by peptide mapping. Materials and methods: rhIFN beta-1b produced by GENERIUM and endoproteinase Glu-C from Staphylococcus aureus V8 were used in the study. The peptide mapping was performed using reverse-phase high-performance liquid chromatography (RP HPLC) and high-resolution mass spectrometry. Statistical evaluation of the results included calculation of the arithmetic mean, standard deviation, and coefficient of variation. Results: the authors developed and certified a Russian Pharmacopoeia reference standard for structural identification of rhIFN beta-1b (PhRS 3.2.00447). The certified characteristic is the range of retention times of characteristic peaks: the absolute retention time was 42.0–43.2 for the third (reference) peak, the relative retention time was 0.61–0.66 for the first peak, 0.68–0.73 for the second peak, 1.04–1.06 for the fourth peak, 1.14–1.15 for the fifth peak, 1.22–1.24 for the sixth peak, and 1.29–1.30 for the seventh peak. Conclusions: the authors developed requirements for the rhIFN beta-1b pharmacopoeial reference standard. The material chosen as the candidate reference standard was an intermediate rhIFN beta-1b product sampled before addition of human serum albumin. The quality control was carried out in accordance with the developed specification. The authors analysed the amino acid sequence of the molecule, confirmed the presence of the disulfi e bond, and obtained the certifi d characteristic of the reference standard. Comparative analysis of the peptide maps of the certified rhIFN beta-1b pharmacopoeial reference standard and the rhIFN beta-1a reference standard revealed differences between the maps, and, therefore, confirmed the relevance of the developed reference standard.

Retracted: Human Interferon Inducible Transmembrane Protein 3 (IFITM3) Inhibits Influenza Virus A Replication and Inflammation by Interacting with ABHD16A.

[This retracts the article DOI: 10.1155/2021/6652147.].

Molecular Docking, Dynamics, and WaterSwap Analysis to Identify Anti-aggregating Agents of Insulin and IFN-β.

There are several challenges in the development, and formulation of biologics, particularly concerning their physical stabilities. The self-assembly of peptides like human insulin and interferon beta (IFN-β) has potential to form aggregates in pharmaceutical formulation. Therefore, it is a significant problem in the manufacturing, storage, and delivery of insulin and IFN-β formulations. Amino acids as aggregation suppressing additives have been used to stabilize proteins during manufacturing and storage. Several changes to the B chain's C-terminus have been proposed in an attempt to improve insulin formulation. The core segments of the A and B chains (SLYQLENY and LVEALYLV) have recently been identified as sheet-forming areas, and their microcrystalline structures have been exploited to construct a high-resolution insulin amyloid fibril model. Here, we have chosen twenty-one amino acids to develop as additives in rendering the insulin and IFN-β aggregations. Thereafter, integrated molecular docking studies of single layer monomers of full-length insulin and IFN-β have been performed to identify structural elements (amino acids) that can act as disaggregating agents. The stability of the best-docked amino acid complexes was judged using molecular dynamics studies. Finally, phenylalanine was identified as a disaggregation agent for insulin, and lysine, tyrosine, phenylalanine, and tryptophan were identified as disaggregation agents for IFN-β from the molecular dynamics study. These findings may open a novel proposal to explore further in vitro studies to increase the stability of the insulin and IFN-β formulation.

Phycocyanin Fusion Constructs for Heterologous Protein Expression Accumulate as Functional Heterohexameric Complexes in Cyanobacteria.

Overexpression of heterologous proteins from plants, bacteria, and human as fusion constructs in cyanobacteria has been documented in the literature. Typically, the heterologous protein "P" of interest is expressed as a fusion with the abundant CpcB β-subunit of phycocyanin (PC), which was placed in the leader sequence position. The working hypothesis for such overexpressions is that CpcB*P fusion proteins somehow accumulate in a soluble and stable form in the cytosol of the cyanobacteria, retaining the activity of the trailing heterologous "P" protein of interest. The present work revealed a substantially different and previously unobvious picture, comprising the following properties of the above-mentioned CpcB*P fusion constructs: (i) the CpcB*P proteins assemble as functional (α,β*P)3CpcG heterohexameric discs, where α is the CpcA α-subunit of PC, β*P is the CpcB*P fusion protein, the asterisk denotes fusion, and CpcG is the 28.9 kDa PC disc linker polypeptide CpcG1. (ii) The (α,β*P)3CpcG1 complexes covalently bind one open tetrapyrrole bilin co-factor per α-subunit and two bilins per β-subunit. (iii) The (α,β*P)3CpcG1 heterohexameric discs are functionally attached to the Synechocystis allophycocyanin (AP) core cylinders and efficiently transfer excitation energy from the assembled (α,β*P)3CpcG1 heterohexamer to the PSII reaction center, enhancing the rate of photochemical charge separation and electron transfer activity in this photosystem. (iv) In addition to the human interferon α-2 and tetanus toxin fragment C tested in this work, we have shown that enzymes such as the plant-origin isoprene synthase, β-phellandrene synthase, geranyl diphosphate synthase, and geranyl linalool synthase are also overexpressed, while retaining their catalytic activity in the respective fusion construct configuration. (v) Folding models for the (α,β*P)3CpcG1 heterohexameric discs showed the recombinant proteins P to be radially oriented with respect to the (α,β)3 compact disc. Elucidation of the fusion construct configuration and function will pave the way for the rational design of fusion constructs harboring and overexpressing multiple proteins of scientific and commercial interest.

Recombinant human erythropoietin and interferon-\u03b2-1b protect against 3-nitropropionic acid-induced neurotoxicity in rats: possible role of JAK/STAT signaling pathway

3-Nitropropionic acid (3-NP) model serves as a beneficial tool to evaluate the effect of novel treatments for Huntington’s disease (HD). The aim of the present study was to demonstrate the neuroprotective effect of recombinant human erythropoietin (rhEPO) and interferon-beta-1b (IFN-β-1b) in 3-NP-induced neurotoxicity in rats. Rats were injected with 3-NP (10 mg/kg/day, i.p) for 2 weeks and were divided into five subgroups; the first served as the HD group, the second received rhEPO (5000 IU/kg/every other day, i.p.) for 2 weeks, the third received rhEPO starting from the 5th day of 3-NP injection, the fourth received IFN-β-1b (300,000 units, every day other day, s.c) for 2 weeks, and the last received IFN-β-1b starting from the 5th day of 3-NP injection. All treatments significantly improved motor and behavior performance of rats. Moreover, all treatments markedly restored mitochondrial function as well as brain-derived neurotrophic factor level, and reduced oxidative stress biomarkers, pro-inflammatory mediators, nuclear factor kappa B expression, caspase-3, and Bax/Bcl2 ratio in the striatum. In conclusion, the present study demonstrates the neuroprotective potential of rhEPO or IFN-β-1b on 3-NP-induced neurotoxicity in rats. Furthermore, our study suggests that activation of JAK2/STAT3 or JAK1/STAT3 may contribute to the neuroprotective activity of rhEPO or IFN-β-1b, respectively. We also found that early treatment with rhEPO did not confer any benefits compared with late rhEPO treatment, while early IFN-β-1b showed a marked significant benefit compared with late IFN-β-1b.

Distinct molecular phenotypes involving several human diseases are induced by IFN-\u03bb3 and IFN-\u03bb4 in monocyte-derived macrophages

Human Interferon (IFN) lambda 3 (IFN-λ3) and IFN-λ4 are closely linked at the IFNL locus and show association with several diseases in genetic studies. Since they are only ~30% identical to each other, to better understand their roles in disease phenotypes, comparative studies are needed. Monocytes are precursors to macrophages (monocyte-derived macrophages; MDMs) that get differentiated under the influence of various immune factors, including IFNs. In a recent study, we characterized lipopolysaccharide-activated M1 and M2-MDMs that were differentiated in presence of IFN-λ3 or IFN-λ4. In this study, we performed transcriptomics on these M1 and M2-MDMs to further understand their molecular phenotypes. We identified over 760 genes that were reciprocally regulated by IFN-λ3 and IFN-λ4, additionally we identified over 240 genes that are significantly affected by IFN-λ4 but not IFN-λ3. We observed that IFN-λ3 was more active in M2-MDMs while IFN-λ4 showed superior response in M1-MDMs. Providing a structural explanation for these functional differences, molecular modeling showed differences in expected interactions of IFN-λ3 and IFN-λ4 with the extracellular domain of IFN-λR1. Further, pathway analysis showed several human infectious diseases and even cancer-related pathways being significantly affected by IFN-λ3 and/or IFN-λ4 in both M1 and M2-MDMs.

&lt;i&gt;In vitro&lt;/i&gt; antiviral activity of VIFERON® rectal suppositories against SARS-CoV-2

In 2020–2021, the world was engulfed by the pandemic of a new coronavirus infection (COVID-19) caused by the SARS-CoV-2 virus. The low population coverage with vaccination against COVID-19 and the lack of herd immunity result in the need to find an effective and safe etiotropic treatment. Medicinal agents for treatment of COVID-19, approved while preparing this publication, have several limitations related to the conditions of their use and/or population category. In this situation, interferon-containing drugs widely used in Russia and the CIS for prevention and treatment of viral infectious diseases, i.e. ARVI and influenza, may hold promise. This study aims to confirm in vitro antiviral activity against SARS-CoV-2 for the preparation VIFERON® containing recombinant human interferon alpha-2b (IFNα-2b). Materials and methods. Vero CCL-81 cells were infected with hCoV-19/StPetersburg-RII3524VR4/2020 strain of SARS-CoV-2 at doses of 10 TCID50 or 100 TCID50 per well. The suppressive effect of IFN-2b, extracted from VIFERON® in dosage form of rectal suppositories, was evaluated by qRT-PCR at 24 h and 48 h after the infection of cells in two schemes, simulating preventive (24 h before infection) and therapeutic (2 h after infection) use of drugs. Results. IFNα-2b at concentrations of 800, 400, 200, 100 and 50 IU/ml, extracted from rectal suppositories of VIFERON®, showed high biological activity, displayed as inhibition of SARS-CoV-2 strain replication in both infectious doses evaluated either at 24 h or at 48 h after cell infection. The “preventive” vs. “therapeutic” scheme was found to be more effective. In the “preventive” scheme the virus titre decreased by more than 3 lg TCID50 at 24 hours post-infection and by 5–6 lg TCID50 at 48 hours post-infection after administration of 800 IU/ml IFNα-2b. Conclusion. The study results evidence that VIFERON® in dosage form of rectal suppositories may be promising for prevention and treatment of new coronavirus infection in clinical practice.

Comparative Study of Commercial Media to Improve GMP Manufacturing of Recombinant Human Interferon β- 1a by CHO Cells in Perfusion Bioreactor

Comparative Study of Commercial Media to Improve GMP Manufacturing of Recombinant Human Interferon β- 1a by CHO Cells in Perfusion Bioreactor

Next-generation version of recombinant human interferon-beta: A molecular dynamic simulation study

The ability of recombinant human interferon beta-1b (IFNβ-1b) to elicit the production of anti-IFNβ-1b antibodies becomes a significant issue for interferon therapy in patients. Mitigating the immunogenicity issues requires understanding the stimulating factors in observed immune responses. Among multiple factors, epitopes might be the main reason for the observed immunogenicity of this drug. In this study, we investigate the ability of epitopes in IFNβ-1b to bind to the MHC class II receptors. We applied point mutations to reduce the binding affinity of the existing epitopes of IFNβ-1b to MHC class II molecules. Several bioinformatics methods were applied to compare the binding affinity of both native and mutated epitopes to the MHC class II receptors. We identified three residues, Tyrosine 3, Tyrosine 132, and Glycine 162, that if altered to Leucine, Leucine, and Glutamate, respectively, leads to a decline in the binding affinity of IFNβ-1 b's epitope to the MHC class II receptors.This suggests that we can decline the binding affinity of therapeutic IFNβ-1b in silico by introducing those point mutations.

Use Intein Cleavable Polyhydroxyalkanoate Synthase Fusions to Improve Protein Solubility.

Recombinant E. coli producing intein-cleavable polyhydroxyalkanoate synthase fusions mediates the intracellular formation of polyhydroxyalkanoate (PHA) particles densely coated with intein-cleavable target protein fusion. These PHA particles can be efficiently purified from lysed cells. The self-cleaving intein performs as a bio-linker between the PHA synthase and the target protein. The tagless target protein can be released as pure soluble protein from the PHA particles by a simple pH reduction to 6.0. Here we describe that PHA particles serve as bioseparation resin for purification of soluble target proteins with pharmaceutical grade purity, similar to commercial affinity separation technologies. This cost-effective technique does not involve multiple complicated protein purification procedures, and we have exploited this approach to purify six target proteins: green fluorescent protein (GFP) from A. victoria, antigen Rv1626 from M. tuberculosis, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).

IN SILICO CHARACTERIZATION OF HUMAN INTERFERON ALPHA/BETA RECEPTOR 2 (ISOFORM A, B AND C) PROTEIN

Objective: To predict the tertiary structure of human interferon alpha/beta receptor 2 protein.&#x0D; Study Design: Structure prediction by using bio informatics tools.&#x0D; Place and Duration of Study: Department of Biochemistry, Swat Medical College (STMC), Saidu Shareef, Swat, Pakistan, from Aug 2019 to Dec 2019.&#x0D; Methodology: All protein sequences of human interferon alpha/beta receptor 2 (isoforma, b and c) (IFNAR-2) were retrieved through the BLAST search (The Basic Local Alignment Search Tool) from available databases ‘NCBI’ (National Centre for Biotechnology Information) and ‘Uni Prot KB’ (The Universal Protein Resource). Sequence alignment was conducted by using Clustal Omega, to get the consensus sequence for IFNAR-2 protein. Consensus protein sequence of human IFNAR-2 was used for the prediction of the three-dimensional structure by employing Swiss-Model Server. Moreover, subcellular localization analysis was also performed by using CELLO2GO program.&#x0D; Results: Structural model of human IFNAR-2 protein was predicted and evaluated by Ramachandran dimension. Cellular localization of tertiary topological domains of the predicted models were revealed probability of localization of IFNAR-2 protein (isoform a, b &amp; c) is highest in the plasma membrane due to the presence of the transmembrane alpha helical regions.&#x0D; Conclusion: This study predicted the tertiary structural dimensions of human IFNAR-2 protein, including the specific topological domains that contribute towards the subcellular compartmentalization and functional characteristics.

Cloning, Transformation and Expression of Human IFN-γ by Genetic Engineering in Saccharomyces cerevisiae

Human interferon-gamma (hIFN-γ) is a Glycoprotein pertinence to a distinct group of interferon, called type II interferons, which have an immunological function to respond to antigenic stimuli such as bacteria, viruses, fungi, and any infections by a microorganism. The hIFN-γ is produced by natural killer T (NKT) and natural killer (NK) cells during the immune response as part of the innate immune response and by Th1 CD4 and CD8 cytotoxic T lymphocytes (CTL) effector T cells upon the development of antigen-specific immunity. Recombinant hIFN-γ has been produced in different expression systems comprising prokaryotic, insect, fungal (yeasts), protozoan, mammalian cells, and plant. In the present study, pET28a plasmid was used in this research for preparation to insert and pYES2 plasmid for cloning and expression of human IFN-γ gene in yeast. The trademark of hIFN-γ has been produced in Escherichia coli is termed ACTIMMUNE®, but the human interferon- gamma was produced in the prokaryotic expression system is unglycosylated form. This result increases in cost due to increased purification, as well as short-life in the bloodstream, but it is biologically active. This study aimed to use Saccharomyces cerevisiae 4741 strain as a eukaryotic system for expression of hIFN-γ cDNA instead of a prokaryotic system in glycosylation patterns as modified translation. But the results are not satisfactory as the produce of yields.

Mammalian sialyltransferases allow efficient Escherichia coli-based production of mucin-type O-glycoproteins but can also transfer Kdo.

The prospect of producing human-like glycoproteins in bacteria is becoming attractive as an alternative to already-established but costly mammalian cell expression systems. We previously described an Escherichia coli expression platform that uses a dual-plasmid approach to produce simple mucin type O-glycoproteins: one plasmid encoding the target protein and another O-glycosylation machinery. Here, we expand the capabilities of our platform to carry out sialylation and demonstrate the high-yielding production of human interferon α2b and human growth hormone bearing mono- and disialylated T-antigen glycans. This is achieved through engineering an E. coli strain to produce CMP-Neu5Ac and introducing various α-2,3- and α-2,6 mammalian or bacterial sialyltransferases into our O-glycosylation operons. We further demonstrate that mammalian sialyltransferases, including porcine ST3Gal1, human ST6GalNAc2 and human ST6GalNAc4, are very effective in vivo and outperform some of the bacterial sialyltransferases tested, including Campylobacter jejuni Cst-I and Cst-II. In the process, we came upon a way of modifying T-Antigen with Kdo, using a previously uncharacterised Kdo-transferase activity of porcine ST3Gal1. Ultimately, the heterologous expression of mammalian sialyltransferases in E. coli shows promise for the further development of bacterial systems in therapeutic glycoprotein production.