The neutralization of interferons by antibody. I. Quantitative and theoretical analyses of the neutralization reaction in different bioassay systems.

The highly specific ability of antibodies to inhibit the biologic activity of cytokines or other therapeutic proteins is widely used in research and a subject of increasing clinical importance. The need exists for a standardized approach to the reporting of neutralizing antibody potency soundly based on theoretical and practical considerations and tested by experimental data. Pursuant to the original studies of Kawade on the theoretical and functional aspects of neutralization of interferons (IFN), experimental data were obtained by different laboratories employing varied methodology to address two hypotheses concerning the nature of IFN neutralization reactions, based on a derived formula that allows expression of neutralizing power as the reduction of 10 laboratory units (LU)/ml to 1 LU/ml, the end point of most bioassays. Two hypotheses are posed: (1) antibody acts to neutralize a fixed amount of biologically active IFN molecules, or (2) antibody reduces IFN activity in a set ratio of added/residual biologically active IFN. The first, or fixed amount, hypothesis relates to the reactivity of high-affinity antibodies neutralizing equimolar amounts of antigen, whereas the second, or constant proportion, hypothesis postulates a reduction in the ratio of total added IFN to residual active IFN molecules, such as a low-affinity antibody might exhibit. Analyses of data of the neutralization of IFN-alpha and IFN-beta are presented, employing human polyclonal antibodies and murine monoclonal antibodies (mAb). The theoretical constructs of Kawade are extended in the Appendix and correlated with new experimental data in the text. The data clearly indicate that the low-antibody affinity, constant proportion hypothesis, rather than the high-antibody affinity, fixed amount hypothesis, is applicable, if the bioassay is sensitive to IFN. The findings presented here and in the following paper (pp. 743-755, this issue) taken together provide the basis for a standardized method of expression of neutralizing potency and substantiate the earlier operational 10/1 LU/ml approach recommended by the World Health Organization. The accompanying paper relates neutralization results to the sensitivity of the bioassay to IFN and describes the rationale for a recommended unit of antibody neutralization.

The neutralization of interferons by antibody. II. Neutralizing antibody unitage and its relationship to bioassay sensitivity: the tenfold reduction unit.

The importance of establishing a common method of reporting neutralizing antibody levels is emphasized by the fact that patients injected repeatedly with a human interferon (HuIFN) may develop such antibodies that can abrogate the beneficial effects of the treatment. The earlier experimental and theoretical constructs of Kawade led to certain recommendations by the World Health Organization (WHO) concerning the methodology of neutralization tests and how to report the resultant data. A WHO international collaborative study on two human sera with antibodies against HuIFN-alpha and HuIFN-beta provided the opportunity not only to test the theoretical concepts concerning the neutralization reaction with data obtained in different bioassay systems in different laboratories but also to obtain enough data points for statistical evaluation with bioassays having a great range of sensitivity to IFN. The analyses substantiate and extend the original conclusions of Kawade that the neutralization follows the reaction mode of low-affinity antibody, in accord with the constant proportion hypothesis by which antibody reduces IFN activity in a set ratio of added/residual biologically active IFN, a consequence of the low molar concentration of free IFN at the neutralization end point. The present results support the recommendation that the preferred way to state the index of neutralization of antibodies is a titer (t), calculated by the formula t = f(n - 1)/9, where f is the reciprocal of the antibody dilution achieving the end point, and n is the IFN concentration measured in that day's titration. The tenfold reduction unit (TRU) of neutralization is proposed for use in expressing the quantity, or unitage, of IFN neutralizing antibody. The utility of its application is explained. The use of the index of neutralization described and the proposed derivative term of antibody unitage, TRU, should help make the results from different laboratories employing different bioassay systems more readily comparable and interpretable, provided the bioassays are sufficiently sensitive to IFN.

Y2, the smallest of the Sendai virus C proteins, is fully capable of both counteracting the antiviral action of interferons and inhibiting viral RNA synthesis.

An open reading frame (ORF) overlapping the amino-terminal portion of the Sendai virus (SeV) P ORF in the +1 frame produces a nested set of carboxy-coterminal proteins, C', C, Y1, and Y2, which are referred to collectively as the C proteins. The C proteins are extremely versatile triple-role players; they counteract the antiviral action of interferons (IFNs), inhibit viral RNA synthesis, and are involved in virus assembly. In this study, we established HeLa cell lines stably expressing the C, Y1, and Y2 proteins individually and examined the capacities of these cells to circumvent the antiviral action of alpha/beta IFN (IFN-alpha/beta) and IFN-gamma and to inhibit viral transcription. The assay protocols included monitoring of IFN-alpha/beta-mediated signaling by interferon-stimulated response element-driven reporter gene expression and of the antiviral state induced by IFN-alpha/beta and IFN-gamma and measurement of reporter gene expression from an SeV minigenome, as well as quantification of SeV primary transcripts. When necessary, the activities measured were carefully normalized to the expression levels of the respective C proteins in cells. The data obtained clearly indicate that the smallest protein, Y2, was as active as the C and Y1 proteins in both counteracting the antiviral action of IFNs and inhibiting viral transcription. The data further show that intracellular transexpression of either C, Y1, or Y2 rendered HeLa cells moderately or only poorly permissive for not only wild-type SeV but also 4C(-) SeV, which expressed none of the four C proteins. On the basis of these findings, the roles of SeV C proteins in the natural life cycle are discussed.

Suppression of interferon-induced antiviral activity in cells expressing hepatitis C virus proteins.

To elucidate the mechanism of the persistent nature of hepatitis C virus (HCV) infection, we examined whether the expression of HCV proteins affect the antiviral activity of interferon (IFN). Antiviral activity of IFN in HepG2 cells expressing all HCV (type 1b) proteins was much lower than vector control (VC) HepG2 cells when encephalomyocarditis virus (EMCV) was used as a challenge virus. Lesser sensitivity to IFN was also observed in cells expressing NS3, NS4, and NS5 and in cells expressing only NS5A. In contrast, HepG2 cells expressing core, E1, E2, NS2, and NS3 proteins were equally sensitive to IFN as VC cells. We then tested the antiviral activity by IFN in two human amnion-derived FL cell lines expressing NS5A from two different clones, one with an intact sequence of IFN sensitivity-determining region (ISDR) and the other with a mutated ISDR sequence. They were almost equally insensitive to IFN treatment when EMCV was challenged. HCV thus has functional protein(s), possibly NS5A, to suppress IFN-induced antiviral activity and plays an important role in virus-cell interaction and regulation of viral replication.

Il-1, EGF, and HGF suppress the antiviral activity of interferon in primary monkey hepatic parenchymal cells.

The purpose of this study was to elucidate the mechanism behind low efficacy of interferon therapy to hepatitis C virus infected patients by using primary monkey hepatic parenchymal cells as a surrogate of primary human liver cells. The effects of various cytokines on the antiviral activity of IFNs in the monkey hepatic cells were studied to search for physiological inhibitors. Interleukin-1 alpha, EGF, and HGF showed suppressive effects on the antiviral activity of IFN-alpha, -beta in primary monkey hepatic cells when examined by the yield reduction method using vesicular stomatitis virus (VSV). In contrast, 50 ng/ml of TNF and IL-6 had no suppressive effect on the IFN-induced antiviral state in the hepatic cells.

Isolation of a measles virus variant: protection of newborn mice from measles encephalitis by 24 h prior intracerebral inoculation with the variant.

A small plaque mutant with reduced neurovirulence in newborn mice was obtained from Edmonston strain measles virus after propagation for 5 months in NIH3T3 cells. It retained the antigenicity of the parental virus and tended to induce higher neutralizing antibody titers in the adult BALB/c mice. The intracerebral (but not intraperitoneal) inoculation of the live mutant virus one day before prevented the newborn BALB/c mice from encephalitis caused by the intracerebral challenge with the parental strain at a dose of 10-20 LD50. The intracerebral inoculation with the mutant virus whose replication capacity was inactivated by UV-irradiation was ineffective. The protection was not attributed to interferons nor to viral interference. The mechanism remains unknown.

Nitric oxide induction by pertussis toxin in mouse spleen cells via gamma interferon.

We examined the major pathogenic substances of Bordetella pertussis for the ability to induce nitric oxide, and important biological function of macrophages, via gamma interferon in spleen cells. B. pertussis, which produces a variety of pathogenic substances, including pertussis toxin and filamentous hemagglutinin, causes a severe respiratory disease. Nitric oxide was detected in the culture fluid of spleen cells stimulated with pertussis toxin or its B oligomer but not in the culture fluid of spleen cells stimulated with the A protomer of pertussis toxin or with filamentous hemagglutinin. Incubation of the peritoneal exudate macrophages with pertussis toxin, B oligomer, A protomer, or filamentous hemagglutinin induced little nitric oxide, whereas incubation with gamma interferon induced a significant amount of nitric oxide. The induction of nitric oxide in spleen cells stimulated with pertussis toxin was completely inhibited by anti-gamma interferon antibody. The treatment of spleen cells with anti-Thy-1.2 antibody plus complement followed by stimulation with pertussis toxin decreased the secretion of gamma interferon and nitric oxide. These results suggest that gamma interferon from T lymphocytes stimulated with pertussis toxin induces nitric oxide.

Enhanced phosphorylation of nuclear 21-kDa and 34-kDa proteins in hepatoma cell death induced by tumor-necrosis factor-alpha.

The role of nuclear protein phosphorylation in intracellular signal transduction of tumor-necrosis factor-alpha (TNF-alpha) in the human hepatoma cell line PLC(PRF/5) was investigated. TNF-alpha, which displays cytolytic activity against PLC hepatoma cells, elevated the in vitro phosphorylation of two nuclear proteins (21 kDa and 34 kDa) 16 h after treatment. The cytotoxicity and enhanced nuclear protein phosphorylation by TNF-alpha treatment decreased in the presence of dexamethasone. Both the 21-kDa and 34-kDa proteins were extracted with 2.2 M NaCl from nuclear pellets and phosphorylated in kinase reaction mixtures containing a high concentration of salt. By phosphoamino acid analysis, the specificity of the nuclear kinase was found to be directed toward serine residues. The protein kinase inhibitors H7, staurosporine and herbimycin A, inhibited the phosphorylation of the 21-kDa and 34-kDa proteins in vitro, but calphostin C and heparin did not. The treatment of cells with 4 beta-phorbol 12-myristate 13-acetate or okadaic acid did not affect the in vitro phosphorylation of the two nuclear proteins. An anti-Fas antibody increased the phosphorylation of the 21-kDa and 34-kDa proteins in PLC cells. DNA fragmentation was observed in PLC cells treated with TNF-alpha and anti-Fas antibody after 24 h treatment. These data suggest an involvement of nuclear protein kinase in signal-transduction pathways of apoptotic cell damage triggered by TNF-alpha in PLC hepatoma cells.

[Current state of national or international standards of interferons and cytokines].

Expert committee on Biological standardization (ECBS) of WHO had asked for an advisory committee for preparation of WHO international cytokine standards because of complexities by the rapid increase of number of cytokine molecules some of which are entered into clinical trials. The new meeting of the WHO informal Consultation on Cytokine standards was started from last spring and going to be held annually. The main mission of the committee was considered to be promoting the use of WHO international cytokine standards and reference materials etc., which must be available world wide. Various standard preparations of cytokines and growth factors are available at the National Institute of Biological Standards and Control (NIBSC) in the United Kingdom, some of which have been accepted by WHO as international standards (Table 1). Japanese national standards which had been prepared before establishment of WHO standards, and being labeled as Japanese reference Units (JRU) have to be calibrated with WHO standards to know International Units (IU). Japanese national references of IL-2 and TNF alpha were calibrated by collaboration with related companies and NIH Japan and the concluded results are obtained as follows. IL-2 (rec. DNA):1JRU = 1 IU TNF alpha (rec. DNA):1JRU = 35 IU

Synthetic oligonucleotides with certain palindromes stimulate interferon production of human peripheral blood lymphocytes in vitro.

We studied the ability of synthetic single-stranded 30-mer oligodeoxyribonucleotides (oligoDNAs) with three different kinds of hexamer palindromic sequence to induce interferon (IFN) production of human peripheral blood lymphocytes (PBL). When PBL was cultured with oligoDNA having a palindrome of AACGTT or GACGTC, IFN activity was detected by bioassay in the culture fluid after 8 h, and the amount of IFN reached the maximum after 18 h. IFN-alpha was predominantly produced, and small amounts of IFN-beta and IFN-gamma were also found. OligoDNA with the palindrome ACCGGT had no effect.

Biological function of recombinant IL-6 expressed in a baculovirus system.

The cDNA of human interleukin-6 (IL-6) was cloned into baculovirus DNA. Insect cells (Spodoptera frugiperda cells) infected with the recombinant baculovirus secreted a large amount of 22K protein into the culture medium. This culture fluid contained high biological activity of growth stimulation of a mouse myeloid cell line (MH-60). The IL-6 was purified by a one-step procedure employing immunoaffinity chromatography of monoclonal antibody to IL-6. The specific activities (BSF-2 reference units/mg protein) of the original culture medium and the purified material from the one-step purification were 0.3-1 x 10(6) and greater than 2 x 10(7), respectively. The highly purified IL-6 could not induce an antiviral state in human diploid fibroblast (FS-4) cells.

An enzyme-linked immunosorbent assay for the measurement of human interleukin-6.

An enzyme-linked immunosorbent assay has been developed to measure human interleukin-6. The assay, based on the avidin-biotin amplified two-step sandwich method, is quick (requiring 4.5 h), sensitive (detecting 9.5 pg/ml) and satisfactory in reproducibility and specificity. It shows good correspondence with the results of bioassays, and it is not affected by serum and plasma components. These results indicate that this ELISA is suitable for application to clinical samples, which is a major advantage over the widely used bioassays.

A temperature-sensitive mutant of Newcastle disease virus defective in intracellular processing of fusion protein.

A temperature-sensitive mutant (ts3) of Newcastle disease virus was physiologically characterized. All major viral structural proteins were synthesized at the permissive (37 degrees C) and nonpermissive (42 degrees C) temperatures, but the fusion (F) glycoprotein was not cleaved at 42 degrees C. In immunocytochemical electron microscopy, the F protein was abundant in the rough endoplasmic reticulum but not in cytoplasmic membrane at 42 degrees C. Noninfectious hemagglutinating virus particles containing all major structural proteins except the F protein were released at 42 degrees C from infected cells. We concluded that the defect in ts3 resides in the intracellular processing of the F protein.

Establishment of strongly neutralizing monoclonal antibody to human interleukin-6 and its epitope analysis.

Three monoclonal antibodies against human interleukin-6 were established and characterized. One antibody was shown to strongly neutralize both the Ig-inducing and hybridoma/plasmacytoma growth activity of interleukin-6. The results of its epitope analysis using protease treated interleukin-6 and immobilized antibody indicated that this neutralizing antibody binds to a peptide corresponding to Leu151-Lys171 of interleukin-6 molecule. Further analysis using synthetic peptides showed that a shorter peptide corresponding to Ala153-Thr162 can also inhibit the binding of the antibody to interleukin-6. These results suggest that this carboxyl-terminal region plays a crucial role in interleukin-6 functions.

Interleukin-1 can inhibit interferon-beta synthesis and its antiviral action: comparison with tumor necrosis factor.

Earlier studies showed that both tumor necrosis factor (TNF) and interleukin-1 (IL1) can inhibit virus replication in cultured cells. However, in human FS-4 fibroblasts, in which recombinant human TNF protected cells from encephalomyocarditis (EMC) virus infection, recombinant human IL1 alpha and IL1 beta failed to induce antiviral protection. Moreover, both forms of IL1 inhibited the development of the TNF-induced antiviral state. To elucidate the mechanism of this inhibition, we examined the effect of IL1 on the synthesis of interferon-beta (IFN-beta), stimulated with polyinosinate.polycytidylate [poly(I).poly(C)]. When added 2 h or more before poly(I).poly(C), both forms of IL1 had a strong inhibitory effect on IFN-beta synthesis, as determined by antiviral assay of the IFN-beta protein or by quantitation of IFN-beta mRNA levels in Northern blot analysis. However, when IL1 was added simultaneously with poly(I).poly(C), or 2 h after poly(I).poly(C), IFN-beta synthesis was increased. The inhibitory action of IL1 on poly(I).poly(C)-induced IFN-beta synthesis was abolished in the presence of cycloheximide, suggesting that it is mediated indirectly by an IL1-induced product in the FS-4 cells. In addition to its ability to inhibit IFN-beta synthesis, IL1 also caused a partial reversal of the antiviral action of IFN-beta. In contrast to IL1, TNF did not inhibit IFN-beta synthesis, nor did it interfere with the antiviral action of IFN-beta. Simultaneous addition of TNF and poly(I).poly(C) to FS-4 cells enhanced IFN-beta synthesis. Under proper conditions TNF and IFN-beta showed an additive antiviral effect.

Dexamethasone inhibits feedback regulation of the mitogenic activity of tumor necrosis factor, interleukin-1, and epidermal growth factor in human fibroblasts.

Tumor necrosis factor (TNF), interleukin-1 (IL-1), and epidermal growth factor (EGF) were mitogenic for human diploid FS-4 fibroblasts. Dexamethasone amplified the growth-stimulating action of all three agents. Amplification of the growth-stimulating action was maximal when dexamethasone was added along with TNF or EGF; no amplification was seen if the addition of dexamethasone was delayed for more than 3 hr. Prolonged simultaneous treatment with TNF and EGF resulted in less growth stimulation than treatment with EGF alone. Dexamethasone abolished this apparent antagonistic interaction between TNF and EGF. Dexamethasone also inhibited the antiviral action of TNF against encephalomyocarditis (EMC) virus in FS-4 cells. TNF and IL-1 increased the steady state level of interferon (IFN)-beta 2 mRNA but failed to induce detectable levels of IFN-beta 1 mRNA in FS-4 cells. Dexamethasone inhibited the increase of IFN-beta 2 mRNA levels by IL-1 or TNF. Inhibition of IFN-beta synthesis is likely to be responsible for the inhibition of the TNF-induced antiviral state by dexamethasone. Since IFNs suppress cell growth, inhibition of endogenous IFN-beta synthesis may also be responsible for the amplification by dexamethasone of the growth-stimulating action of TNF and IL-1. Amplification of the mitogenic action of EGF by dexamethasone appears to be mediated by different mechanism.

Mitogenic effect of double-stranded RNA in human fibroblasts: role of autogenous interferon.

The synthetic double-stranded RNA polyinosinate-polycytidylate [poly(I).poly(C)] was mitogenic in cultures of human foreskin fibroblasts, as demonstrated by a stimulation of 3H-thymidine incorporation and an increase in cell density. Poly(I).poly(C) is a potent inducer of interferon (IFN)-beta in human fibroblasts. Single-stranded poly(l) or poly(C) were not mitogenic in human fibroblasts and did not stimulate IFN production. Antiserum to interferon (IFN)-beta, added to poly(I).poly(C)-stimulated cultures in order to neutralize endogenously generated IFN, markedly amplified the mitogenic action. Under similar experimental conditions, antiserum to IFN-beta did not enhance the mitogenic action of epidermal growth factor (EGF). Dexamethasone enhanced the mitogenic action of poly(I).poly(C) in a manner similar to antiserum against IFN-beta. This effect of dexamethasone correlated with its marked inhibitory action on poly(I).poly(C)-stimulated IFN production. Together with the results of other related studies, these findings support the notion of an evolutionary link between the generation of a mitogenic signal and IFN induction. In addition, these results support the concept that autocrine secretion of IFN-beta can exert negative feedback control of cell proliferation.

Protective effect of antibodies to two viral envelope glycoproteins on lethal infection with Newcastle disease virus.

The protective effect of humoral immunity against lethal infection of chickens with Newcastle disease virus was studied. Chickens hatched from eggs laid by hens vaccinated with live attenuated Newcastle disease virus vaccine possessed antibody to various components of the virus, and were resistant to a challenge with a virulent strain of Newcastle disease virus which was 100 per cent fatal for the offspring of nonvaccinated hens. Passive administration of antiserum raised against whole virions provided susceptible chickens protection comparable to that seen in the birds with maternal antibody. When administered passively, both anti-HN serum with virus neutralizing activity, and anti-F serum with only marginal virus neutralizing activity significantly prolonged the survival of infected birds but failed to achieve the level of protection as afforded by the anti-whole NDV serum. The protection provided by the simultaneous presence of anti-HN and anti-F serum was significantly greater than that afforded by either alone and comparable to that of anti-whole NDV serum, indicating the complementary effect of anti-HN and anti-F antibodies not only in cell cultures as reported previously (19), but also in a natural host.

Tumor necrosis factor: receptor binding and mitogenic action in fibroblasts.

Until about two years ago, the only known function of tumor necrosis factor (TNF) was inhibition of tumor growth. Since then it has become apparent that many types of normal and transformed cells express specific high-affinity TNF receptors (Kd 200 pM) and that the presence of receptors does not correlate with susceptibility to the cytotoxic/cytostatic action of TNF. Recent evidence shows that TNF exerts a variety of other important biological activities on cells in culture and in the intact organism. Among the newly recognized activities is a potent mitogenic effect in fibroblasts. Many of the activities of TNF overlap the actions of interleukin-1 (IL-1).