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Antimicrobial Agents and Chemotherapy, September 2005, p. 3770-3775, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3770-3775.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Quantification of Different Human Alpha Interferon Subtypes and Pegylated Interferon Activities by Measuring MxA Promoter Activation

Catherine François,¹ Isabelle Bernard,¹ Sandrine Castelain,¹ Bryan Charleston,² Martin D. Fray,² Jean-Claude Capiod,³ and Gilles Duverlie^1*

Laboratory of Virology, Centre Hospitalier Universitaire of Amiens, Amiens, France,¹ Institute for Animal Health, Compton Laboratory, Compton, Newbury, Berkshire RG20 7NN, United Kingdom,² Laboratory of Hematology, Centre Hospitalier Universitaire of Amiens, Amiens, France³

Received 4 February 2005/ Returned for modification 12 April 2005/ Accepted 16 June 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alpha interferons (-IFNs) are potent biologically active proteins synthesized and secreted by somatic cells during viral infection. Quantification of -IFN concentrations in biological samples is used for diagnosis. More recently, recombinant IFNs have been used as antiviral, antiproliferative, and immunomodulatory therapeutic agents, and particularly for the treatment of chronic hepatitis C virus infection. For this purpose, IFN has recently been coupled to polyethylene glycol (PEG) to improve the pharmacokinetic properties. The measure of -IFN in biological samples from treated patients could be useful to ensure compliance to therapy and the true IFN activity in relation to viral decay during follow-up. In particular, it could be used to monitor the PEG-IFN concentration in patients treated for hepatitis C virus infection. The most frequently used test is a bioassay based on the antiviral property of the IFN, but the assay is not highly reproducible. Here, we present a reporter test based on MxA promoter activation of chloramphenicol acetyltransferase expression (Mx-CAT). MxA is an antiviral protein induced and tightly regulated by -IFN. The Mx-CAT assay showed good reproducibility of 15% and was suitable to quantify PEG-IFN and numerous other -IFN subtypes as well, despite a differential MxA promoter activation in relation with the subtype. A good correlation was obtained with the reporter assay and a commercial enzyme-linked immunosorbent assay on samples from treated patients. This test could be useful for monitoring IFN therapy of chronically infected hepatitis C virus-infected patients treated with the standard IFN, PEG-IFN, and probably forthcoming recombinant IFNs.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interferons (IFNs) are the major antiviral cytokines produced during a viral infection. IFNs and ß are produced by numerous cell types after viral infection. Type II IFN is represented by IFN-, also called immunological interferon, as it is implicated in the immunological response. The presence of IFN in biological samples is frequently used to identify viral infection (9).

IFN-/ß binds to its specific receptor and activates the Janus kinases Jak1 and Tyk2, leading to tyrosine phosphorylation of STAT proteins. These proteins multimerize and translocate to the nucleus to activate a large number of IFN-stimulated genes, whose protein products mediate the pleiotropic effects of IFN (8). There are several hundred genes transcriptionally regulated by IFN (3). Among the major effector gene products are the double-stranded RNA-dependent protein kinase (PKR), the 2'-5' oligoadenylate synthetase (2-5OAS), and the MxA protein, of which the antiviral activities have been well characterized (12).

The human genome encodes two related Mx proteins, MxA and MxB, that are induced by IFN-/ß (12). The MxA protein comprises 662 amino acids, whereas the MxB protein is made of 715 amino acids (1) and they are GTPases that belong to the superfamily of dynamin-like GTPases (13). MxB has not been found to display an antiviral activity (6). MxA protein is the best characterized of the known IFN-inducible gene products with antiviral activity. It had been established that MxA protein alone was sufficient to block the replication of some viruses, for example, orthomyxoviruses, in the absence of any other IFN-inducible proteins (6).

Analysis of the promoter region of the MxA gene revealed three potential IFN-stimulated response elements (ISRE), but only the two proximal elements were functional. It also contains three putative class II interleukin-6 responsive elements, one putative NF-B binding site, and one Sp1 binding site that may provide means of interaction with the basal transcriptional machinery (10).

Using bovine cells it has been shown that within 3 h of exposure to IFN, measurable amounts of bovine MxA protein can be detected in the cytoplasm, with gene transcription continuing for at least 24 h (4). In the absence of an IFN stimulus, bovine cells do not synthesize measurable amounts of Mx protein.

Several techniques have been used to measure the IFN concentration in biological samples. The reference method used the vesicular stomatitis virus (VSV) challenge bioassay that permits the detection of IFN in biological samples by measuring its antiviral activity against VSV (9). Other recently described methods of titrating IFN concentrations are based on an enzyme-linked immunosorbent assay (ELISA) or on inhibition of the hepatitis C virus (HCV) replicon (14).

IFN- is also used as the therapeutic agent for the treatment of chronic HCV infection. Recently, new formulations based on pegylation permitted a better response to IFN treatment (18). Pegylation is the association between IFN and polyethylene glycol (PEG) that modifies the pharmacokinetics and immunological properties to permit once-weekly injection and a better antiviral effect which correlates with the sustained induction of IFN-stimulated genes.

Here we present the evaluation of a bioassay based on Madin-Darby bovine kidney (MDBK) cells expressing the human MxA promoter linked to a chloramphenicol acetyltransferase (CAT) reporter gene (5). This assay was used to measure IFN-/ß in biological samples from HCV-infected patients treated with recombinant human IFN or pegylated IFN. In contrast to an ELISA-based assay, the bioassay permits the measurement of the biologically active IFN in the sample and avoids interference from the presence of antibodies against IFN. The MxA reporter assay could also allow better reproducibility than the antiviral bioassay currently used.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell line. MDBK-t2 cells were obtained from Martin D. Fray and were utilized for measuring bovine IFN (5). These cells expressed the MxA promoter linked to a CAT reporter gene. They were grown in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Cergy Pontoise, France) containing 4,500 mg/liter of glucose, GlutaMAX I, and pyruvate and supplemented with 10% of fetal calf serum and 10 mg/ml of blasticidin (Invitrogen, Cergy Pontoise, France). Cells were passed twice per week at a dilution of 1:16, depending on the cell growth, by using trypsin-EDTA (Invitrogen, Cergy Pontoise, France).

Interferons. Recombinant IFN- 2a (PBL biomedical Laboratories, Piscataway) and PEG-IFN- 2a (Pegasys, Roche, Neuilly sur Seine, France) were used as a standard to establish the linear range of the Mx-CAT bioassay for IFN- 2a and PEG-IFN- 2a, respectively.

PEG-IFN- 2b (PEG-Intron, Schering-Plough, Levallois-Perret, France) was used as a standard to evaluate the reproducibility of the bioassay and for the in vivo application.

Other recombinant IFNs- were obtained from the human IFN sampler (PBL Biomedical Laboratories, Piscataway, NJ).

These standard samples were diluted and were frozen in DMEM containing 10% of fetal calf serum at –80°C until use. They were used to establish the standard curves of IFN in each assay.

Mx-CAT assay protocol. For each assay, MDBK-t2 cells were plated at a density of 10⁶ per well in six-well plates. After 18 h of incubation at 37°C in 5% CO₂, the culture medium was discarded and replaced with 1 ml of DMEM with 10% fetal bovine serum and 100 µl of sample or standard. The standards were obtained by serial twofold dilution of known amounts of the different IFNs used in this work (100 UI/ml for IFN-2a, 9 ng/ml for PEG-IFN-2a, and 625 pg/ml for PEG IFN-2b). The cells were cultured for an additional 24 h at 37°C in 5% CO₂.

CAT expression was determined using a commercial enzyme-linked immunosorbent assay kit (Roche Diagnostics, Meylan, France). In brief, all cells were lysed for 20 min in 500 µl of lysis buffer. A 200-µl aliquot of the lysate was added to individual wells of the CAT-ELISA 96-well plate and assayed according to the manufacturer's instructions. The potencies of the unknown samples were calculated from a standard curve and constructed from 1:2 serial dilution of the different standards.

Interferon sampler. The human IFN- sampler (PBL Biomedical laboratories) includes 2.10⁵ units/ml of each species of IFN- (IFN-2, IFN-8, IFN-10, IFN-1, IFN-21, IFN-5, IFN-14, IFN-17, IFN-7, IFN-6, IFN-4 and IFN-16). Fifty units of each IFN- were used to evaluate the MxA promoter activation. One hundred microliters of each IFN were tested on the MDBK-t2 cells as described above. A CAT enzyme standard dilution curve was prepared to quantify the MxA promoter activation. This curve was plotted in accordance to the manufacturer's instructions. For each test, the CAT production was measured and was compared to IFN- standard curve.

Measurement of IFN- by ELISA. An IFN- titration was performed using the Human Interferon Alpha ELISA kit (PBL Biomedical Laboratories, Piscataway). One hundred microliters of sample or standard were placed on the precoated microtiter plate in duplicate and incubated for 1 hour at room temperature. After the first incubation and washing, the antibody solution was incubated at room temperature for one hour. After washing, the horseradish peroxidase conjugate solution was added and was incubated for one hour at room temperature. The substrate was added and the reaction was stopped within 15 min. The absorbance at 450 nm was read within 5 min.

A calibration curve (IU/ml) was plotted to calculate the concentration of each sample following the manufacturer's instructions.

In vivo-derived samples. To test the in vivo application of the assay on human samples, the IFN concentration was measured in patients chronically infected by HCV and treated with PEG-IFN-2b in combination with ribavirin as recommended by the European consensus of hepatitis C therapy. The serum samples were collected at different times after the first injection and stored at –80°C until assayed for the IFN activity in either the Mx-CAT reporter gene assay or the human IFN- ELISA kit.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Limit of detection. The analytical sensitivity of the test was evaluated for standard IFN-2a and for both pegylated IFNs. Thus, 100 µl of serial diluted samples of each interferon have been tested on the MDBK-t2 cells as described in Materials and Methods. The limit of detection was 3 units per ml for the standard IFN- 2a. The linearity of the method was obtained between 3 and 100 units per ml for the standard IFN (Fig. 1A) (R² = 0.99, P < 0.001). For the PEG-IFN-2a and PEG-IFN-2b, the linearity was obtained between 1.13 and 9 ng/ml (R² = 0.94, P < 0.01 for PEG-IFN-2a) (Fig. 1B) and 19.5 and 625 pg/ml (R² = 0.99, P < 0,01 for PEG-IFN-2b), respectively (Fig. 1C).

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FIG. 1. Linearity of the Mx-CAT reporter assay. Standard IFN-2a (A), PEG-IFN-2a (B), and PEG-IFN-2b (C) were serially diluted to obtain standard samples; 100 µl of each sample was incubated on MDBK-t2 cells. After 24 h of incubation, cells were lysed and the CAT activity was determined by an ELISA CAT assay. The optical density (OD) was measured at 405 nm against 490 nm and is reported on the graph depending on the IFN concentration. The coefficient of regression (R2) indicates the linearity of detection. PEG-IFN concentrations were expressed in ng/ml and pg/ml for PEG-IFN-2a and PEG-IFN-2b, respectively, and in international units/ml for IFN-2a as indicated by the manufacturer. The statistical regression analysis was realized with StatView.

Assay reproducibility. The reproducibility of the assay was estimated for PEG-IFN-2b. Ten replicates of two samples (156.2 pg/ml and 625 pg/ml) were analyzed in the same assay to determine the intra-assay reproducibility. The intra-assay coefficients of variation were 8.6% (range: 141 to 192 pg/ml, standard deviation 15.65) and 7.2% (range: 520 to 672 pg/ml, standard deviation 46.46) for the low and the high control, respectively (Fig. 2A).

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FIG. 2. Reproducibility of the Mx-CAT reporter assay. The reproducibility of the technique was studied for PEG-IFN-2b. Two samples (156.2 and 625 pg/ml) were analyzed 10 times in the same assay (intra-assay variability, A) and in 10 different assays (interassay variability, B). The points represent the mean and the bars represent the standard deviation. The statistical analysis was realized with StatView.

The interassay reproducibility was estimated by analyzing both samples in ten different assays. The interassay coefficients of variation were, respectively 14.4% (range: 135.1 to 228.4 pg/ml, standard deviation 27.33) and 9.4% (range: 503 to 583 pg/ml, standard deviation 53.15) for the low and the high control, respectively (Fig. 2B).

Evaluation of MxA promoter activation by different IFNs-. To evaluate the capacity of the test to measure different IFNs-, we have used the human IFN- sampler containing twelve subtypes of IFN- as described in Materials and Methods. The bio-activities of these IFNs were determined by the manufacturer using the cytopathic effect inhibition assay as previously described (11). Fifty units of each sample of IFN were tested on MDBK-t2 cells. The MxA promoter activity was measured by quantifying the CAT expression. The standard curve of the CAT enzyme was realized in duplicate (Fig. 3A). This test was realized for each IFN- and the optical density obtained for each subtype was reported on the CAT standard curve. Each subtype of IFN- was detectable with this test. Nevertheless, the MxA promoter activity was different for each IFN. Three groups could be identified compared to IFN-2a induction: IFN-7 and IFN-6 were equal inducers of MxA. IFN-17 was two times better an inducer than IFN-2a. The others were poorer inducers than IFN-2a.

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FIG. 3. Ability of the Mx-CAT assay to detect different IFN- subtypes. Twelve subtypes of IFN- were tested on the Mx-CAT reporter assay; 50 units of each IFN was incubated with MDBK-t2 cells and CAT quantity was determined after 24 h. The quantity of CAT was determined with a standard enzyme (A). For each IFN, the quantity of CAT was calculated and is reported on the graph (B). Each stick represents the mean CAT quantity and the error bars represent one standard deviation.

PEG-IFN titration in patients with chronic hepatitis C. To evaluate the IFN activity in patients chronically infected by HCV and treated with the combination of PEG-IFN-2b and ribavirin, PEG-IFN concentrations were measured in the serum during the treatment. The same samples were analyzed in parallel with an ELISA immunological test to compare these two methods.

The results are summarized in Fig. 4. The coefficient of correlation was 0.93 (P < 0.001) between the two techniques. The IFN concentrations were in general a little higher for the cellular test based on the CAT activity than the immunological one (Fig. 4). The PEG-IFN concentrations were included between 19.5 and 700 pg/ml during the follow-up.

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FIG. 4. Comparison of immunological method and CAT assay on HCV patients treated with PEG-IFN--2b. 60 samples of HCV patients were analyzed by the IFN ELISA and the Mx-CAT reporter test. Each IFN concentration is reported on the graph and the coefficient of correlation was calculated. The PEG-IFN--2b concentrations are expressed in pg/ml. The statistical correlation study was realized with Excel.

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we have evaluated a bioassay to quantify different IFNs in biological samples. This bioassay was not based on the protection of cultured cells against viral cytopathic effect, but on the activation of the MxA promoter by IFN. The MxA promoter containing two functional IFN-responsive elements was placed upstream of the CAT reporter. As the basal level of Mx proteins is low and is strictly controlled by IFN, it has been a good candidate as a marker for the presence of IFN. The limit of detection of the test is 3 UI/ml for the standard human IFN as for the bioassay using the VSV challenge bioassay that is the reference method.

The VSV challenge method had been utilized recently to measure PEG-IFN in patient sera with a reproducibility of 30% (2). The majority of the assay based on cell cultures was often carried out in 96-well microtitration plates. However, nonuniformity of 96-well microtitration plates and variable accuracy of multichannel micropipettes can introduce untoward biases which can contribute to uneven responses across the plates. The Mx-CAT test was realized on six-well plates with 100 µl of samples that certainly reduced the variations. The critical point was the number of cells. The assay was based on the expression of an IFN-inducible reporter gene in cell culture. The overall quantity of reporter expressed depends of the number of cells in each well. It would be the same in each well in the assay and in each assay. This could explain that the interassay variability (14.4%) was higher than the intra-assay variability (8.6%).

The Mx-CAT reporter test was done on MDBK cells. These bovine cells are currently used to realize the IFN bioassay virus challenge as they are very sensitive to human IFN-/ß.

The interpretation of the results was not a subjective method as that utilized for the challenge virus assay. In the Mx-CAT protocol, the MxA promoter activation was measured exactly with an ELISA assay. No dilution of the samples was necessary for the measurement of the PEG-IFN concentrations of treated HCV patients since the linear range of the technique comprised the values obtained in this assay during IFN therapy.

The therapeutic IFNs were well measured with the Mx-CAT bioassay with a good correlation between the measured titers and the sample titers.

To determine the extent of induction of MxA by different IFN- subtypes, we used the IFN- sampler that contains 12 IFN- subtypes. The results show a good detection of all the subtypes but equivalent antiviral activity (50 IU/ml) did not result in equivalent induction of the MxA promoter. Indeed, IFN-8 was not a good activator of the MxA promoter. IFN-8 is one of the major subtypes present in the clinically applied IFN- preparation derived from the lymphoblastoid cell line BALL-1. The combination of IFN-8 and IFN-2 had shown a synergistic antiviral activity in the hepatocellular carcinoma cell line HepG2 (17). IFN-8 was also observed as the most potent inhibitor of viral cytopathic effects and this antiviral activity was well correlated with the 2-5OAS activation. IFN-2, -5, and -10 had an intermediate activity and IFN-1 had a little antiviral activity on the liver cell lines (15).

Here, we have found that IFN-8 was not a good inducer of the MxA promoter activation. It is possible that bovine cells or kidney cells were perhaps not as sensitive as the human liver cell lines to IFN-8. However, another explanation is the IFN8 antiviral activity described above was studied with Sindbis virus and vesicular stomatitis virus and should be mediated by proteins other than MxA, such as PKR or/and 2-5O AS. In contrast, IFN-1 had a poor antiviral activity against VSV and Sindbis virus on hepatocellular cell lines and was a good activator of the MxA promoter. IFN-17 seems to be the greater MxA inducer and was described as a good 2-5OAS inducer too (16). These data suggest that the biologic activities of different IFN- subtypes could correlate with their respective binding affinities to the cells used or that the signal transduction could be modulated according to the cell types and their receptors.

From a clinical point of view, the biological assay presented here was very useful to monitor IFN-2 therapy. A correlation between the drug concentration in the serum and the treatment efficacy has been suggested. This test could be used to monitor the therapeutic follow-up and compliance to treatment as for human immunodeficiency virus therapy. Adaptation of the dose of IFN could be proposed to improve the treatment efficacy.

However, differences between subtypes should be kept in mind as clinical trials using new recombinant IFN are ongoing. When available, their capacity to induce the MxA promoter would have to be tested to use appropriately the Mx-CAT reporter assay presented here.

We have also compared the Mx-CAT assay to those from an IFN ELISA on chronically infected patients treated with the combination PEG-IFN and ribavirin. There was a good correlation between the two assays (R² = 0.94, P < 0.001), but the values obtained with the bioassay were often higher than those obtained with the ELISA. The MxA promoter contained other elements of activation like NF-B or interleukin-6-responsive elements. The difference observed could be due to the presence of other cytokines in the biological samples that could interfere with the IFN transactivation. Another explanation could be that the PEG moiety interferes with the epitopic recognition of anti-IFN antibodies used in the immunological test. Furthermore, measuring biological activity could be more sensitive and relevant than measuring protein concentration.

This bioassay is more reproducible than the VSV challenge assay usually used, but the cost is higher since our bioassay utilized an ELISA to quantify the CAT expression. Using the ELISA probably allowed a better reproducibility since the interpretation of the results was less subjective. Furthermore, this assay did not use infectious system, which renders it easier to set. A direct ELISA against IFN would be the best solution but these tests were generally less sensitive when measuring IFN in patient samples. This may be due to certain inhibitors such as heterophile antibodies or rheumatoid factors (7). The HCV replicon-based IFN assay is a good way to directly show the antiviral activity of IFN (14). This assay utilizes an HCV replicon expressing the firefly luciferase protein. However, the results were difficult to interpret since the relation between the decrease of the HCV replicon and the IFN concentration is not linear.

Neutralizing IFN antibodies IFN have been involved in some IFN therapy failures. The bioassays were the only test permitting us to show IFN-neutralizing antibodies.

As shown above, the Mx-CAT bioassay could be used for the follow up of patients chronically infected with HCV and treated with PEG-IFN. The linearity of the technique permits us to measure IFN in biological samples easily. Several IFN- subtypes could be detected using the Mx-CAT method, and the sensitivity was equivalent to that of the bioassay for standard IFN. It would be also interesting to test that technique on biological samples to measure IFN induced during viral infection.

 
This work was supported by the Programme Hospitalier de Recherche Clinique de Picardie (PHRC, 2001).

 
* Corresponding author. Mailing address: Faculté de Médecine et de Pharmacie, Laboratoire de Virologie, 3 rue des Louvels, 80000 Amiens, France. Phone: 33 3 22 82 79 17. Fax: 33 3 22 82 79 28. E-mail: gilles.duverlie{at}u-picardie.fr.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Antimicrobial Agents and Chemotherapy, September 2005, p. 3770-3775, Vol. 49, No. 9
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.9.3770-3775.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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