Evaluation of the Anti-Viral Activity of Human Recombinant Interferon Lambda-1 against SARS-CoV-2.

The antiviral activity of recombinant human IFN-lambda type 1 (IFNλ-1) against culture strain of SARS-CoV-2 virus was determined by infecting a highly sensitive VeroE6 coronavirus cell culture after preincubation test (the cell monolayer was incubated with 4-fold dilutions of IFNλ-1 in a concentration range of 0.16-42,500 ng/ml in a culture medium for 12 h at 37°C) and without preincubation (simultaneous addition of different concentrations of IFNλ-1 and SARS-CoV-2 infection in a dose of 102 TCID50). The created recombinant human IFNλ-1 demonstrated obvious antiviral activity against SARS-CoV-2 virus in vitro. In the tests with and without preincubation, IFNλ-1 exhibited significant activity, although somewhat lower in variant with simultaneous addition of IFNλ-1 and virus to the cell culture. It should be noted that the antiviral effect of IFNλ-1 was observed in a wide range of concentrations.

Broad-Spectrum Robust Direct Bactericidal Activity of Fish IFNφ1 Reveals an Antimicrobial Peptide-like Function for Type I IFNs in Vertebrates.

Type I IFNs (IFN-Is) play pivotal roles in host defense against viral infections but remain enigmatic against bacterial pathogens. In this study, we recombinantly expressed and purified intact grass carp (Ctenopharyngodon idella) IFNφ1 (gcIFNφ1), a teleost IFN-I. gcIFNφ1 widely powerfully directly kills both Gram-negative and Gram-positive bacteria in a dose-dependent manner. gcIFNφ1 binds to LPS or peptidoglycan and provokes bacterial membrane depolarization and disruption, resulting in bacterial death. Furthermore, gcIFNφ1 can efficiently protect zebrafish against Aeromonas hydrophila infection and significantly reduce the bacterial loads in tissues by an infection model. In addition, we wonder whether antibacterial IFN-I members exist in other vertebrates. The amino acid compositions of representative IFN-Is with strong positive charges from Pisces, Amphibia, reptiles, Aves, and Mammalia demonstrate high similarities with those of 2237 reported cationic antimicrobial peptides in antimicrobial peptide database. Recombinant intact representative IFN-I members from the nonmammalian sect exhibit potent broad-spectrum robust bactericidal activity through bacterial membrane depolarization; in contrast, the bactericidal activity is very weak from mammalian IFN-Is. The findings display a broad-spectrum potent direct antimicrobial function for IFN-Is, to our knowledge previously unknown. The results highlight that IFN-Is are important and robust in host defense against bacterial pathogens, and unify direct antibacterial and indirect antiviral bifunction in nonmammalian jawed vertebrates.

Interferon-mediated reprogramming of membrane cholesterol to evade bacterial toxins.

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.

Expression and purification of biologically active bovine Interferon λ3 (IL28B) in Pichia pastoris.

Interferon lambda-3 (IFNλ3) which is also known as IL28B is a member of type III Interferons which are structurally and genetically different from type I Interferons. These Interferons induce signal transduction pathways similar to type I Interferons which results in the activation of Interferon Stimulated Genes (ISGs). This group of Interferons are tissue specific and reported to have antiviral activity. In the present communication, we report the expression of bovine IFNλ3 gene (coding for the mature protein) in Pichia pastoris, purification of the expressed protein and evaluation of its biological activity. About 19 kDa protein expressed by the transformed Pichia cells, secreted into the media and the protein was purified by SP-Sepharose ion exchange chromatography with NaCl stepwise gradient elution. Specificity of the protein was confirmed by Western blotting. Pichia expressed IFNλ3 was found to be biologically active, as it induced ISGs (Mx protein, OAS and PKR genes) in bovine PBMCs. Further it was also found to modulate Th1/Th2 cytokines expression in the stimulated bovine PBMCs.

Soluble expression and one-step purification of recombinant mouse interferon-λ3 in Escherichia coli.

Interferon (IFN)-λ3, a member of the type III IFN family, is a pleiotropic cytokine that exhibits potent antiproliferative, antiviral, and immunoregulatory activities. For further functional study of IFN-λ3, we developed an efficient procedure that includes cloning, expression, and purification to obtain relatively large quantity of mouse IFN-λ3 fusion protein. The mature IFN-λ3 protein-coding region was cloned into the prokaryotic expression vector pET-44. IFN-λ3 contains a hexahistidine tag at its C-terminus. We used Ni(2+)-nitrilotriacetic acid agarose-affinity chromatography to purify the expressed soluble protein. The purified IFN-λ3 inhibited significantly IL-13 production in stimulated RAW264.7 macrophages. Our findings show that the production of soluble IFN-λ3 proteins by the pET-44 vector in Escherichia coli is a good alternative for the production of native IFN-λ3 and could be useful for the production of other IFN proteins.

Biological effects of chicken type III interferon on expression of interferon-stimulated genes in chickens: comparison with type I and type II interferons.

Interferons (IFNs) are key mediators that activate host defense mechanisms against viruses. The recently identified mammalian Type III IFN has biological effects similar to type I IFN. However, the biological effects of type III IFN have not yet been characterized in birds. We compared the effects of chicken type III IFN (IFN-λ) with type I (IFN-ß) and type II (IFN-γ) IFNs on IFN-stimulated genes (ISGs) using recombinant proteins expressed in Escherichia coli. Recombinant chicken IFN-λ inhibited influenza virus replication and induced the mRNA expression of the ISGs, Mx and OAS, in chicken embryonic fibroblasts (CEFs) in a dose-dependent manner. However, the effective dose of IFN-λ was higher than that of IFN-ß and IFN-γ. Furthermore, the effect of IFN-λ on induction of Mx and OAS was lesser than that of IFN-ß, but comparable to that of IFN-γ. These results indicate that chicken IFN-λ has the potential to induce ISGs and inhibit viral replication in chicken cells.

Intron-containing type I and type III IFN coexist in amphibians: refuting the concept that a retroposition event gave rise to type I IFNs.

Type I and III IFNs are structurally related cytokines with similar antiviral functions. They have different genomic organizations and bind to distinct receptor complexes. It has been vigorously debated whether the recently identified intron containing IFN genes in fish and amphibians belong to the type I or III IFN family or diverged from a common ancestral gene, that subsequently gave rise to both types. In this report, we have identified intron containing type III IFN genes that are tandemly linked in the Xenopus tropicalis genome and hence demonstrate for the first time that intron containing type I and III genes diverged relatively early in vertebrate evolution, and at least by the appearance of early tetrapods, a transition period when vertebrates migrated from an aquatic environment to land. Our data also suggest that the intronless type I IFN genes seen in reptiles, birds, and mammals have originated from a type I IFN transcript via a retroposition event that led to the disappearance of intron-containing type I IFN genes in modern vertebrates. In vivo and in vitro studies in this paper show that the Xenopus type III IFNs and their cognate receptor are ubiquitously expressed in tissues and primary splenocytes and can be upregulated by stimulation with synthetic double-stranded RNA, suggesting they are involved in antiviral defense in amphibians.

Interferons, interferon-like cytokines, and their receptors.

Recombinant interferon-alpha (IFN-alpha) was approved by regulatory agencies in many countries in 1986. As the first biotherapeutic approved, IFN-alpha paved the way for the development of many other cytokines and growth factors. Nevertheless, understanding the functions of the multitude of human IFNs and IFN-like cytokines has just touched the surface. This review summarizes the history of the purification of human IFNs and the key aspects of our current state of knowledge of human IFN genes, proteins, and receptors. All the known IFNs and IFN-like cytokines are described [IFN-alpha, IFN-beta, IFN-epsilon, IFN-kappa, IFN-omega, IFN-delta, IFN-tau, IFN-gamma, limitin, interleukin-28A (IL-28A), IL-28B, and IL-29] as well as their receptors and signal transduction pathways. The biological activities and clinical applications of the proteins are discussed. An extensive section on the evolution of these molecules provides some new insights into the development of these proteins as major elements of innate immunity. The overall structure of the IFNs is put into perspective in relation to their receptors and functions.

High yield expression, refolding, and characterization of recombinant interferon alpha2/alpha8 hybrids in Escherichia coli.

Interferons (IFNs) are a family of pleiotropic cytokines used for the treatment of various viral infections and cancers. The low-cost production of IFNs with high biological value and the discovery of IFNs with improved properties are important for the treatment of these diseases as well as for understanding the physiological functions of these compounds. We describe a protein expression system for the production of IFNs alpha2, alpha8, and their hybrids in insoluble form in Escherichia coli, coupled to an efficient two-step optimized refolding and histidine-tag purification protocol. The expressed IFNs were of high biological value, as shown in antiviral and antiproliferative assays and some had specific activities higher than those of the commercially available interferon preparations and exhibited novel properties. This time-efficient, optimized protein expression method allows for the production of not just a single interferon subtype but several native and hybrid IFNs with relatively high yield and low cost that can be used in functional and potentially clinical assays.

Detection of squirrel monkey retroviral sequences in interferon samples.

BACKGROUND/AIMS: Interferons have been used therapeutically in viral infections, and as immunomodulants in the treatment of different types of cancers. Interferons have been prepared from human lymphoid cell-lines, such as Namalwa, that contain integrated copies of squirrel monkey retrovirus proviral DNA. Squirrel monkey retrovirus is related to simian type D retroviruses, such as Mason-Pfizer monkey virus. Thus it is important to determine if these retroviral sequences are present in interferon preparations purified from human cell lines. METHODS: DNA samples were prepared from 75 commercial interferon preparations and analyzed for squirrel monkey retrovirus sequences by polymerase chain reaction and DNA sequencing. Since single polymerase chain reaction is not as sensitive, a nested polymerase chain reaction strategy was devised in order to detect squirrel monkey retrovirus-pol sequences. Amplification of beta-actin (human) sequences was used to confirm that samples contained human genomic DNA. To determine the authenticity of squirrel monkey retrovirus sequences, we analyzed amplified products by Southern blot hybridization and direct DNA sequencing. RESULTS/CONCLUSIONS: Thirty-nine samples were positive for squirrel monkey retrovirus-pol sequences by nested polymerase chain reaction. It is noteworthy that 29 samples were either weakly or very weakly positive by single polymerase chain reaction, thus stressing the importance of our sensitive polymerase chain reaction assay. However, it remains to be determined whether the residual DNA sequences detected by our sensitive nested polymerase chain reaction assay have biological consequences.

[Natural interferons: the methods of their isolation, the properties and characteristics of the preparations and their use in animal infections]. / Pryrodni interferony: sposoby oderzhannia, vlastyvosti ta osoblyvosti diï preparativ, ïkh zastosuvannia pry infektsiiakh tvaryn.

Data of studies carried out in the field of interferonology during the last 20 years which are based on the proposed new biotechnologies for production of natural alpha- and gamma-interferons (IFNs) have been analyzed. "Know-how" of these technologies consists in the usage of non-traditional source (spleen instead of blood) for preparing the suspension of immunocytes--producers of preparations, in introduction of the stage of "coinduction" when producing alpha-IFNs, use of bacterial lectins of apathogenic bacilli as inducers of gamma-IFN genesis. It was shown, that in vitro and in vivo the both types of preparations several times increased both absorbing and bactericidal activity of phagocytes of swine and cattle peripheral blood, 3-5 times increased antibody genesis in animals after their immunization by colibacteriosis vaccine. It was proved that immunomodulating ability of alpha-IFNs is significantly (3-5 times) higher than that for gamma-IFNs. In non-purified preparations of IFNs the fractions have been discovered which had antibacterial and antitoxic action of wide spectrum of specificity. Both preparations were effective for prophylaxis and therapy of not only viral, but also bacterial infections in animals: transmissive gastroenteritis of swine, parainfluenza of calves, colibacteriosis of pigs and calves, etc., and promoted the increase of animals productivity by 20-35%. If under hemoblastosis alpha-IFN caused remission, they could be effectively treated using homologous gamma-IFN.

Purification of an atypical bovine interferon induced in response to heat shock in bovine cultured cells. Characterization in comparison with the classical alpha, beta and gamma interferon.

Heat-shock induced factor (HSIF) was excreted by bovine MDBK cells during their recovery period after a heat shock. This factor has the capacity to induce 2',5' oligoadenylate synthetase activity, an enzyme generally by interferon treatment (J Biol Chem (1987) 262, 4806-4811). We have observed that an antiviral state was also produced in response to heat shock. HSIF was purified 10,000-fold and different techniques showed a copurification of both activities. Certain properties of HSIF were established, such as its molecular mass (45 kDa) and isoelectric point (6.8). This cytokine was acid-sensitive as IFN gamma (type II) and temperature labile contrarily to alpha, beta and gamma bovine IFN. Immunoprecipitation and comparative chromatography on lectines or polynucleotides established that HSIF was structurally different from the three classes of bovine IFN. Moreover, two-dimensional electrophoresis and comparative analysis of [35S] methionine-labeled proteins induced by HSIF alpha, beta or gamma bovine IFN showed that HSIF induces a specific set of proteins. Taken together, all these results strongly suggest that HSIF is a new atypical bovine interferon induced in response to heat shock.

Purification of biological molecules by continuous flow electrophoresis in the Second International Microgravity Laboratory.

The RAMSES instrument is a continuous-flow electrophoresis device that performs separation and purification of biological materials on a preparative scale. It was flown on board the shuttle Columbia during the Second International Microgravity Laboratory mission that took place from 8 to 23 July 1994. This project had a technological aim to qualify the design and the technologies used, and a more scientific one to improve understanding of the phenomena involved in this process. To reach these goals, experiments were performed in which different samples and a variety of operating conditions were used, some of which cannot be applied on earth. Useful purifications were obtained, allowing samples to be collected for further analysis on the ground. These results confirm the predictions of a numerical model that could be used for future process optimization and the samples were found to have retained their biological activity.

Cytopathic effect inhibition assay for canine interferon activity.

Conditions for cytopathic effect (CPE) inhibition assay of canine interferon (IFN) activity in Madin-Darby canine kidney (MDCK) cells and in canine tumor cell line A72 was investigated using the New Jersey strain of vesicular stomatitis virus (VSV). The culture supernatant from canine splenocytes stimulated with ultraviolet-irradiated Newcastle disease viruses was used as reference IFN. MDCK cells were resistant for growth of VSV when the cells were confluent. Full CPE was observed only in a sparsely growing culture. Canine IFN activity could be assayed on less than 10(4) MDCK cells/well of a 96-well microplate, and more than 10(5) TCID50/ml of VSV was required. In A72 cells, VSV growth was not as dependent on cell density as in MDCK cells, requiring 10(3) TCID50/ml of VSV. MDCK-VSV system showed a higher IFN sensitivity than A72-VSV, whereas reproducibility was higher for the latter than the former. Based on these findings, A72-VSV system for canine IFN assay is recommended for practical use due to its easy handling characteristics.

Recombinant duck interferon: a new reagent for studying the mode of interferon action against hepatitis B virus.

Although interferon is widely used to treat chronic hepatitis B virus infections, its mode of action against hepadnaviruses is largely unknown. This deficit is due mainly to the lack of suitable model systems. The duck system could not be used because purified duck interferon was not available in sufficient quantities. We have now cloned a DNA fragment that contains an intronless gene for duck interferon. The primary translation product consists of 191 amino acids, the N-terminal 30 residues of which constitute a signal peptide. Mature duck interferon is 50% identical to the recently cloned chicken interferon. Sequence homology to mammalian interferons is marginal, but conservation of four cysteine residues and inducibility by virus indicate a distant relationship between duck interferon and mammalian type I interferons. Purified recombinant duck interferon from Escherichia coli is biologically active: it activates the interferon-inducible Mx gene, prevents cell destruction by cytolytic RNA viruses, and has a strong inhibitory effect on duck hepatitis B virus in cultured primary duck hepatocytes. This new reagent should help to define the interferon-sensitive step of the hepadnavirus life cycle. Furthermore, the duck system can now be used for systematic studies of the in vivo effectiveness of interferon in chronic hepatitis B virus infections.