Recombinant Human Interferon Alpha-2a Research Articles

Effect of biphasic temperature regime on therapeutic recombinant protein production in the green alga Chlamydomonas reinhardtii

Microalgae are increasingly being considered for recombinant protein production because of low cultivation costs, absence of endotoxins and insusceptibility to human infectious agents. Despite these advantages, the yield of recombinant protein produced in microalgae is still low compared to more established expression systems and optimization at the genetic and cultivation levels is required for this new system to be economically viable. This study investigates the effect of biphasic temperature regimes on the yield of recombinant human interferon alpha 2a (IFN-α2a), a therapeutic protein known for its anti-cancer and anti-viral properties, produced by the model green alga Chlamydomonas reinhardtii (Cr.IFN-α2a). Biphasic growth is commonly employed to increase recombinant protein production in mammalian cell lines used for commercial production of therapeutic proteins, with a lowering of the temperature resulting in higher yields. In this study, lowering the temperature from 25 °C to 15 °C in mid-exponential growth phase increased the accumulation of Cr.IFN-α2a by 3.3-fold while it slowed down the growth of the three C. reinhardtii transgenic lines tested. In contrast, a rise of temperature from 25 °C to 35 °C accelerated cell growth, while negatively impacting the production of Cr.IFN-α2a. After a two-step chromatography purification, the Cr.IFN-α2a produced was estimated to be 53% pure with a yield of 90 μg/L of culture. The amino acid sequence of Cr.IFN-α2a was confirmed by mass spectrometry. However, the anti-viral activity of Cr.IFN-α2a was found to be 10 times lower than the human IFN-α2a standard produced using E. coli when challenged in a cytopathic effect (CPE) assay, likely due to the formation of aggregates. While the molecular mechanisms driving the accumulation of Cr.IFN-α2a at lower temperature remains unclear, our results support that reducing the temperature at the peak of expression is a valid strategy to increase the yield of recombinant Cr.IFN-α2a in C. reinhardtii.

Purification and Stability Expression of Open Reading Frames encoding Fusion and Non Fusion Human Interferon Alpha-2a produced in Methilotropic Yeast Pichia pastoris

Recombinant human interferon alpha-2a (hIFNα-2a) is therapeutic protein that widely used in hepatitis B/C and several cancer treatments. We developed higher molecular weight of hIFNα-2a to improve protein pharmacokinetic profile. The protein was designed as a fusion protein with human serum albumin as protein tag. The protein was produced in Pichia pastoris with 85 kDa in size. This research was aimed to purify, characterize and determine the stability expression of open reading frame (ORF) encoding Fusion and Non fusion forms of hIFNα-2a. The proteins were purified using affinity chromatography and characterized using SDS PAGE and Western Blotting methods. Protein recovery yield was determined by ELISA. Stability expression was applied in generation time until 90th generation. The results showed that the Fusion and Non fusion proteins were successfully purified with 74-79% of protein recovery. The proteins can be recognized by specific monoclonal antibody and verified as hIFNα-2a Fusion and Non fusion with 85 kDa and 19 kDa in size respectively. The expression stability showed that the proteins were still produced in Pichia pastoris until 90th generation time with no significant difference of expression level. To conclude, the expression level of ORFs encoding Fusion and Non fusion hIFNα-2a was stable until 90th generations.

Optimization of Expression Condition, Two Dimensional And Melting Point-Based Characterization of Recombinant Human Interferon Alpha-2a Fusion and Non Fusion Forms

Recombinant Human Interferon Alpha-2a (rhIFNα-2a) is a therapeutic protein that used in hepatitis and cancer treatments. In our previous research, we developed higher molecular weight of the protein through human serum albumin fusion. The fusion and non fusion form of rhIFNα-2a were produced in Pichia pastoriswith 86 kDa and 19 kDa in size respectively. In previous research, protein yield was not reproducible due to unoptimized expression conditions. This reseach was aimed to optimize expression condition process and to characterize the fusion and non fusion forms of rhIFNα-2a. The parameters to observe in overproduction include nutrient (media and methanol concentration) and non nutrient (temperature andincubation period). Affinity and size exclusion cromatographicwere compared in protein purification. BCA assay was used to determine quantity of protein. Protein characterization was conducted using two-dimensional SDS PAGE and denaturation analyses. The optimal condition of expression was achieved using complex media with 1% of methanol for 3 day incubation period at 25°C. The protein yield was reproducible and higher comparing to previous research. Affinity chromatography resulted in higher purity of the proteins comparing to size exclusions. Characterization using two dimensional gel analysis revealed that isoelectric point of rhIFNα-2a is 6.5 for fusion form and 6.0 for non fusion form. The melting points of fusion protein were 56°C and 62°C whilst that of non fusion was 56°C.

Overproduction, characterization and preliminary antiproliferative activity determination of non-tagged recombinant human interferon alpha-2a produced in Pichia pastoris

Herawati N, Wardiana A, Ningrum RA. 2017. Overproduction, characterization and antiproliferative activity determination of non-tagged recombinant human interferon alpha-2a produced in Pichia pastoris. Nusantara Bioscience 9: 97-101. Recombinant human interferon alpha-2a (rhIFNα-2a) has been widely used for antiviral, anticancer as well as immunomodulatory clinical therapy. In our previous research, we constructed the Open Reading Frame (ORF) encoding synthetic rhIFNα-2a to be in framed with N-terminal alpha factor secretion system in pPICZαB shuttle vector. This research covered overproduction, characterization of the protein and preliminary determination of biological potency assay by using the estrogen positive cell line MCF-7. To overproduce the protein, cultivation was performed in Buffered complex medium containing glycerol (BMGY) for 24 h and production was performed in Buffered complex medium containing methanol (BMMY) during 48 hours at 30°C. The recombinant protein was purified by affinity chromatography using blue sepharose resin. Analysis of amino acid sequence by using MS/MS2 mass spectrometry covered 21% of the total amino acid residues. To determine the initial biological activity, the effect of rhIFNα-2a on MCF-7 breast cancer cell line was studied in vitro. The anti-proliferative activity of hIFNα-2a was performed by 3-[4.5-dimethyltiazol-2il]-2.5 diphenylltetrazolium bromide (MTT) assay. The result showed that the protein is able to inhibit MCF-7 proliferation and the reduction of cell growth was dose-dependently. To monitor the protein production, we performed stability expression assay as well. The result informed that the expression of ORF still stable until 60th generation

Improved detection of variants in recombinant human interferon alpha-2a products by reverse-phase high-performance liquid chromatography on a core–shell stationary phase

The detection of variants is one of the important aspects in quality control of recombinant DNA drugs. In this study, a gradient reverse-phase high-performance liquid chromatography (RP-HPLC) method with fluorescence detection is described for the separation of interferon alpha-2a (rhIFN α-2a) from several product related variants. The methodology employed a core–shell C18 column with a linear gradient elution of 0.2% (v/v) trifluoroacetic acid (TFA)-acetonitrile (ACN) at 1.0mL/min, and the temperature of the column was maintained at 60°C. The method was validated in terms of linearity, sensitivity, intra- and inter-day variations. Compared to the European Pharmacopeia RP-HPLC method of rhIFN α-2a analysis, this new method can separate N-methionylated variant in both drug substance and finished product, and analyze the variants in untreated, oxidized sample and slightly degraded samples more efficiently. In conclusion the method has an improved capability to detect variants in rhIFN α-2a products.

The ETR (End Treatment Response) of Chinese Brand Human recombinant interferon alpha 2a plus Ribavirin in chronic hepatitis C patients selected from Combined Military Hospitals in Pakistan

This Study was carried out to determine the End of Treatment Response (ETR) of a Chinese Source Human Recombinant Interferon Alpha 2a and Ribavirin in chronic hepatitis C affected patients, selected from different combined military hospitals in Pakistan. In this study hepatitis affected noncirrhotic patients from different combined military hospitals in Pakistan were selected from September 2010 to April 2011. Sixty seven patients were selected based on the inclusion exclusion criteria. Fifty seven patients completed the 24 weeks therapy for Hepatitis C. The Patients (n=57) had persistently raised serum aminotransferase (ALT), positive HCV antibodies by 3rd generation ELISA, positive HCV RNA by polymerase chain reaction. Study group patients underwent 24 weeks therapy with a Chinese source Interferon and Ribavirin and subsequently followed up for the End of Treatment Response by the assay of HCV RNA by polymerase chain reaction at 24 weeks. Patients of chronic hepatitis C (44 males and 13 females) had age range 20-56 years. After 24 weeks of therapy with a Chinese source Interferon and Ribavirin, 73.7% patients showed a favourable ETR manifested by Negative HCV RNA polymerase chain reaction.Treatment with a Chinese source Interferon and Ribavirin combination therapy for 24 weeks produces a favourable End of Treatment Response in patients of chronic hepatitis C from different military hospitals in Pakistan.

Refolding of Recombinant Human Interferon α-2a fromEscherichia coliby Urea Gradient Size Exclusion Chromatography

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon alpha-2a (rhLFNalpha-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhLFNalpha-2a would pass along the 8.0-3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 x 10(8) IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhLFNalpha-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhl FNalpha-2a refolding in terms of specific activity and mass recovery.

Pegylated human interferon alpha 2a does not induce depression-associated changes in mice

Interferon (IFN) alpha proteins are proinflammatory cytokines having immunomodulating and antiviral properties. States during which cytokine systems are activated (e.g., during viral infection or during treatment of chronic hepatitis C and various malignancies with IFN alpha, etc.) can be associated with depression-like syndromes or even full-blown depressive episodes. Therefore, the role of IFN alpha and other cytokines in the pathogenesis of depressive disorder (“cytokine hypothesis of depression”) has been assessed for many years with contradictory results. We have investigated whether intraperitoneal administration of high doses (up to 600 µg/kg body weight) of pegylated, recombinant human IFN alpha 2a in mice induces changes known to be associated with depression using three different readouts: behavior in a model of despair (Porsolt swim test), presence of anhedonia (sucrose preference test), and sensitivity of the hypothalamic–pituitary–adrenal system (dexamethasone suppression test). We also assessed potential IFN-induced changes in gene expression in the liver. In none of the performed experiments, depression-associated effects could be found despite very high serum levels of IFN-induced antiviral activity compared to levels measured in hepatitis C virus (HCV) patients treated routinely with pegylated recombinant human IFN alpha 2a. The lack of such expected effects is probably due to the fact that pegylated human recombinant IFN alpha 2a does not activate the murine class I IFN receptor. Our results do not support the hypothesis that administration of recombinant pegylated human IFN alpha to mice produces a robust model of depression.

Crystallization and preliminary X-ray diffraction analysis of the complex between a human anti-interferon antibody fragment and human interferon alpha-2A.

Recombinant human interferon alpha-2A (rhIFN-alpha-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic monoclonal antibody (MEDI545; IgG1/kappa) which targets several human interferon alpha subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 134.82, b = 153.26, c = 163.49 A. The diffraction of the crystals extended to 3.0 A resolution. The asymmetric unit contained two Fab-rhIFN-alpha-2A complexes. This corresponded to a crystal volume per protein weight (V(M)) of 3.02 A(3) Da(-1) and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity.

Use of Recombinant Human Interferon Alpha-2a in the Management of a Dog With Epitheliotropic Lymphoma

An 8-year-old, mixed-breed dog with preputial epitheliotropic lymphoma was initially treated with cyclophosphamide, vincristine, and prednisolone. A short-term partial response was followed by disease progression after 4 weeks. Recombinant human interferon alpha-2a was administered starting at week 7. The interferon therapy resulted in rapid resolution of clinical signs and a 10-week disease-free interval. The lymphoma recurred at 17 weeks and did not respond to rescue chemotherapy. Additional oral lesions were treated with localized radiotherapy followed by increased dosages of interferon. This additional interferon treatment resulted in another 12 weeks of stable disease.

P004 Changes in thymic recent output function in patients with acute myeloid leukemia

The influence of recombinant human interferon alpha 2a (rIFN alpha), recombinant human interferon beta 1 (rIFN beta), and recombinant human interferon gamma (rIFN gamma) on human dermal microvascular endothelial cells (HDMEC) cultured in vitro was studied in various rIFN concentrations (0.1 lU/ml-104 IU/ml) over 2,3,4,6,8, and 10 d. Cell morphology and ultrastructure, cell proliferation, expression of class II alloantigens (HLA-DR and HLA-DQ), and intercellular adhesion molecule-1 (ICAM-1) were investigated using an in vitro technique established in our laboratory. All rIFN tested induced alterations of typical HDMEC morphology; the cells became spindle-shaped and fibroblastoid, although they maintained their endothelial cell marker expression. Also, all IFN dose- and time-dependently inhibited the proliferation of HDMEC in vitro (rIFN alpha> beta> gamma), whereby rIFN alpha exerted the strongest growth-inhibitory effect. Alkaline phosphatase anti-alkaline phosphatase (APAAP) immunocytochemistry of the cultured cells showed dose- and time-dependent stimulation of ICAM-1 and class II antigen expression only by rIFN gamma (HLA-DR> HLA-DQ), rIFN alpha and beta did not exert any immunomodulatory activity on HDMEC in vitro. These results indicate that HDMEC are an important target for the action of IFN. Besides growth inhibition, it seems that rIFN gamma in particular may be involved in the modulation of leucocyte adhesion and trafficking by altering the immunophenotype of the endothelial cell population.

Influence of human recombinant interferon-alpha2a (rhIFN-alpha2a) on altered lymphocyte subpopulations and monocytes in Behcet's disease.

In Behçet's disease (BD), several abnormalities of lymphocyte subpopulations have been described. Standard treatment comprises immunosuppressive drugs. We successfully treated 50 patients with ocular BD with interferon-alpha2a (IFN-alpha2a) (response rate 92%), although this is counterintuitive because IFN-alpha is immunostimulatory and can sometimes even induce autoimmune diseases such as systemic lupus erythematosus or rheumatoid arthritis. The aim of the present study was to elucidate the immunomodulatory effects that IFN-alpha might exert on peripheral blood mononuclear cells (PBMC) in BD by examining changes in the distribution of lymphocyte subpopulations under IFN-alpha2a treatment. Fourteen patients with ocular BD were evaluated before and at weeks 4 and 24 of IFN-alpha treatment and compared with 10 healthy controls. PBMC were stained with monoclonal antibodies and measured by flow cytometry. Compared with the controls there is a significant elevation of monocytes (CD14(+)), CD8(+)/gammadelta T cells, CD3(+)/gammadelta T cells, natural killer (NK) cells (CD56(+)/CD16(+)) and activated/regulatory T cells (CD4(+)/CD25(+) and CD8(+)/CD25(+)) in patients with active BD before treatment with IFN-alpha2a. Numbers of naïve T cells (CD8(+)/CD45(+)RA(+)/RO(-), CD4(+)/CD45(+)RA(+)/RO(-)) were significantly lower. Under therapy, NK cells, CD8(+)/gammadelta T cells and CD3(+)/gammadelta T cells decreased significantly, whereas B cells increased. The previously reduced expression of HLA class I on monocytes in HLA-B51-positive patients rose to levels comparable to HLA-B51-negative patients. These results implicate the participation of NK cells and gammadelta T cells, especially CD8(+)/gammadelta T cells, in the pathogenesis of BD and may explain one mechanism by which IFN-alpha2a exerts therapeutic effects. Alternatively, they may result indirectly from remission induction by IFN-alpha2a. The reduced expression of HLA class I on monocytes in HLA-B*51-positive patients might reflect an impaired expression of and antigen presentation by HLA-B*51.

Effect of vacuum drying on protein-mannitol interactions: the physical state of mannitol and protein structure in the dried state.

The purpose of the present studies was to systematically investigate protein-mannitol interactions using vacuum drying, to obtain a better understanding of the effect of protein/mannitol wt/wt ratios on the physical state of mannitol and protein secondary structure in the dried state. Solutions containing beta-lactoglobulin (betaLg):mannitol (1:1-1:15 wt/wt) were vacuum dried at 5 degrees C under 3000 mTorr of pressure. The physical state of mannitol was studied using x-ray powder diffractometry (XRPD), polarized light microscopy (PLM), Fourier-transform infrared (FTIR) spectroscopy, and modulated differential scanning calorimetry (MDSC). XRPD studies indicated that mannitol remained amorphous up to 1:5 wt/wt betaLg:mannitol ratio, whereas PLM showed the presence of crystals of mannitol in all dried samples except for the 1:1 wt/wt betaLg:mannitol dried sample. FTIR studies indicated that a small proportion of crystalline mannitol was present along with the amorphous mannitol in dried samples at lower (less than 1:5 wt/wt) betaLg:mannitol ratios. The T(g) of the dried 1:1 wt/wt betaLg:mannitol sample was observed at 33.4 degrees C in MDSC studies, which indicated that at least a part of mannitol co-existed with protein in a single amorphous phase. Evaluation of the crystallization exotherms indicated that irrespective of the betaLg:protein wt/wt ratio in the initial sample, the protein to amorphous mannitol ratio was below 1:1 wt/wt in all dried samples. Second-derivative FTIR studies on dried betaLg and recombinant human interferon alpha-2a samples showed that mannitol affected protein secondary structure to a varying degree depending on the overall mannitol content in the dried sample and the type of protein.

Temperature- and pH-induced multiple partially unfolded states of recombinant human interferon-alpha2a: possible implications in protein stability.

To study the effect of solution conditions on the structural conformation of recombinant human interferon-alpha2a (IFNalpha2a) to investigate its tendency to form partially unfolded intermediates. The structural properties of IFNalpha2a were studied at various pH values (2.0-7.4) and temperatures (5 degrees C-80 degrees C) using Trp fluorescence emission, fluorescence quenching, near- and far-UV circular dichroism (CD) spectroscopy, and DSC. Fluorescence intensity measurements as a function of temperature indicated the onset of the thermal unfolding of IFNalpha2a, denoted by Td, around 60 degrees C above pH 4.0. Td was not observed at pH 3.5 and below. Acrylamide and iodide quenching studies indicated partial unfolding of protein with decrease in pH and with increase in temperature up to 50 degrees C. Near-UV CD studies indicated a significant loss in the tertiary structure of protein on increase in temperature from 15 degrees C to 50 degrees C at all solution pHs. DSC scans supported results obtained from fluorescence and CD studies at pH 4.0 and below. DSC, however, was insensitive to changes that occurred at moderate temperatures at pH 5.0 and 7.4. IFNalpha2a has a tendency to acquire multiple partially unfolded states with structural conformations sensitive to solution pH and temperature. These states were formed at moderate temperatures, and it is speculated that these partially unfolded states could play an important role in the aggregation of proteins during the long-term storage of aqueous protein formulations.

New and modified interferon alfas: preclinical and clinical data.

Recombinant human interferon (IFN)-alpha was the first biotherapeutic agent approved by the US Food and Drug Administration for the treatment of a human malignancy. Its efficacy has also been demonstrated for treatment of several viral diseases. The human genome codes for 12 IFN-alpha proteins, with IFN alpha-1 and IFN alpha-2 accounting for the majority of the naturally occurring IFN-as. However, only subspecies of IFN alpha-2, recombinant human IFN alpha-2a and IFN alpha-2b, are commercially available in the United States. Other species of IFN-a may demonstrate equivalent or improved efficacy and have more tolerable side effects. This article describes ongoing preclinical and clinical studies of several new and modified IFN-alphas. A current phase I trial of a human recombinant IFN alpha-1 is described. Basic pharmacokinetics and clinical studies of polyethylene glycol (PEG) IFN alpha-2b are reviewed as well. Lastly, two novel types of IFN-a, one gene shuffled and one hybridized with human albumin, are summarized.

Recombinant human interferon alpha-2a therapy in children with chronic immune thrombocytopenic purpura.

In a prospective study, 11 children with chronic immune thrombocytopenic purpura between ages 3 and 18 years were treated with recombinant human interferon alpha 2a (rhIFN alpha-2a). A dose of 3 x 10(6) U/m2 three times weekly for 4 to 5 weeks (one cycle) was administered. Patients were treated with one to four cycles of rhIFN alpha-2a, and the outcomes were measured initially and 18 to 30 months after the last cycle. Good therapeutic responses (defined as platelet count >100 x 10(9)/L) lasting for 18 to 30 months from the last interferon cycle were achieved in 6 of the 11 (55%) patients, including one with a probable spontaneous remission. Fair responses (platelet count 31-60 x 10(9)/L) for 18 months were achieved in 3 of the 11 (27%) patients. Only two patients, each treated only with one interferon cycle, exhibited no response. Side effects of treatment included fever and a flu-like syndrome, which were usually present during the first 14 days of therapy only. Interferon-alpha appears to be an effective therapeutic approach to children with chronic immune thrombocytopenic purpura, with the potential of sustained long-term remission. A randomized, placebo-controlled study is needed to confirm its role in this population.

IFN-beta induces serine phosphorylation of Stat-1 in Ewing's sarcoma cells and mediates apoptosis via induction of IRF-1 and activation of caspase-7.

Four human cell lines derived from Ewing's sarcoma, EW-7, EW-1, COH and ORS, were investigated to establish the effects of human recombinant interferon-alpha2a and human recombinant interferon-beta on cell proliferation and apoptosis. All four cell lines were much more sensitive to the antiproliferative effects of IFN-beta than of IFN-alpha. Analysis of the early signals triggered by IFN-alpha and IFN-beta demonstrated that the two IFNs were similarly effective in inducing tyrosine phosphorylation of the Jak-1 and Tyk-2 kinases and the transcription factors Stat-1 and Stat-2. Interestingly, an additional rapid phosphorylation of Stat-1 on serine was observed after IFN-beta treatment, with concomitant activation of p38 mitogen-activated protein kinase. In these cells, Stat-1 Ser727 phosphorylation in response to IFN-beta was found to be impaired by p38 MAPkinase inhibitor (SB203580). IFN-beta induced the formation of the Interferon Stimulated Gene Factor 3 complex more efficiently than IFN-alpha, as well as sustained induction of IRF-1, which may account for its greater induction of 2'5'oligo(A)synthetase and greater inhibition of cell proliferation. IFN-beta, but not IFN-alpha, induced apoptosis in wild-type p53 EW-7 and COH cell lines, but not in the mutated p53 EW-1 or ORS cell lines. The apoptosis induced by IFN-beta in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activation of caspase-7. Ectopic expression of IRF-1 induced apoptosis in all four cell lines which correlated with the activation of caspase-7 and with the downregulation of the Bcl-2 oncoprotein, as observed for IFN-beta-induced apoptosis in parental EW-7 and COH cell lines.

Lack of p21waf1 and p27kip1 protein induction by interferon-alpha2a in human melanoma cell lines.

The ability of human recombinant interferon-alpha2a (IFNalpha2a) to induce the expression of cyclin-dependent kinase inhibitors p21waf1 and p27kip1 consequent to signal transducers and activators of transcription (STAT) protein activation was investigated in six human melanoma cell lines with different susceptibilities to the antiproliferative effect of the cytokine. All the cell lines expressed IFNalpha and IFNalpha/beta receptors. Exposure for 24 h to IFNalpha2a markedly enhanced the nuclear expression of STAT1 and STAT2 proteins in all the cell lines. However, no induction of p21waf1 or p27kip1 was consistently observed. Overall, results from the study suggest that the induction of such cyclin-dependent kinase inhibitors is not a major mechanism for the antiproliferative effect of IFNalpha2a, at least in human melanoma cell lines.

Synergistic Anti-Varicella-Zoster Virus Activity of Interferon-ά2a and Acyclovir in Corneal Cells

Human corneal stromal (HCS) cells in cultures established from donor corneas can serve as host cells for replication of varicella-zoster virus (VZV). Comparative infectious centers assays suggest that HCS cells are more restrictive hosts than MRC-5 cells, a line of human embryonic lung fibroblasts, commonly used for VZV isolation. VZV propagated in MRC-5 cells was infectious for both MRC-5 and HCS cells, but titers in HCS were one fifth of those in MRC-5 cells. Inhibition of VZV replication in HCS cells by acyclovir (ACV), recombinant human interferon-alpha 2a (IFN-alpha 2a), or the combination of these two antivirals was detected by both an infectious centers-plaque-reduction assay and an ELISA method using monoclonal anti-VZV antibody. In the infectious centers-plaque-reduction assay combinations of ACV with IFN-alpha 2a showed synergistic anti-VZV activity. VZV protein synthesis, as detected by the ELISA, was a less sensitive measure of the antiviral effects producing an ED50 value of 50 microM for ACV, five to ten times the ED50 determined in the plaque reduction assay. In the ELISA, high titers of IFN-alpha 2a (2000 IU/ml) decreased virus antigen expression only slightly, while combinations of ACV and IFN-alpha 2a were synergistic in their detected anti-VZV activity. These data document the replication of VZV in HCS cells and demonstrate that VZV is sensitive to the synergistic antiviral action of combinations of IFN-alpha 2a and ACV.

Combined effects of interferon-alpha2a and 13-cis-retinoic acid on human melanoma cell growth and STAT protein expression.

We assessed the antiproliferative effects of human recombinant interferon-alpha2a (IFNalpha2a) and 13-cis-retinoic acid (13cis-RA) on two human melanoma cell lines (JR8 and M14). Both cell lines showed a very modest sensitivity to IFNalpha2a and 13cis-RA as single agents. In JR8 cells, the combination of the two compounds consistently produced simple additive effects. In contrast, different effects of the combination were recorded in the M14 cell line depending on the treatment schedule. Specifically, an additive interaction was observed when IFNalpha2a and 13cis-RA were given in sequence, independently of the order of drug administration, whereas a supra-additive antiproliferative effect was seen when cells were simultaneously exposed to the two drugs. Exposure to 1000 IU/ml IFNalpha2a markedly increased the nuclear expression of signal transducers and activators of transcription (STAT) proteins in both cell lines. By itself 10 microM 13cis-RA did not affect STAT protein expression or modify the extent of activation of such proteins by IFNalpha2a. Results from our study showed an enhancement of the antiproliferative activity of IFNalpha2a and 13cis-RA when given in combination and suggest that such an enhancement is not mediated by a concomitant effect of 13cis-RA on STAT protein activation.