Direct antiproliferative effects of recombinant human interferon-alpha B/D hybrids on human tumor cell lines.

The purpose of these studies was to examine the antiproliferative properties of 16 recombinant human IFN-alpha B/D hybrids against various human tumor lines of different histological origin and to determine whether any of the hybrid molecules possessed immunomodulating activity that could active antitumor properties in peripheral blood monocytes of normal donors. Hybrids with the B domain at the NH2 terminal end exhibited higher activity for antiviral activity and a higher level of direct antitumor antiproliferative activities as compared with hybrids with the D domain at the NH2 terminal end. The positive hybrids were directly cytostatic to melanoma, glioblastoma, renal carcinoma, colon carcinoma, and prostatic carcinoma cells. Tumor cell sensitivity to IFN-alpha hybrids was independent of sensitivity to IFN-gamma or to Adriamycin. The growth of a normal cell line (human embryo fibroblast) was unaffected by IFN-alpha hybrids but was completely arrested by Adriamycin. Some of the IFN-alpha hybrids were also cytostatic to mouse melanoma, lung carcinoma, and fibrosarcoma cell lines, albeit at lower levels than they were to human cells. The incubation of monocytes with IFN-alpha hybrids with the B domain at the NH2 terminal end was also associated with marked antitumor cytotoxicity. Kinetic studies, however, indicated that this activity was attributable to IFN-alpha carried on monocytes and acting directly on tumor cells. We conclude that recombinant human IFN-alpha B/D hybrids possess potent direct antiproliferative activity against a large variety of human tumor lines.

Interferon-alpha hybrids.

The alpha-interferons (IFN-alpha) belong to a family of polypeptides comprising several subtypes. Using recombinant DNA technology, it has been possible to create IFN hybrids that provide novel combinations of the amino acid residues from the parental protein sequences. They have been used to study structure-activity relationships of IFN-alpha and interactions with the IFN-alpha receptor, and to create analogs of natural IFNs with novel properties for potential therapeutic application. The biological data obtained with these hybrids are now evaluated in terms of the published structural and homology models of IFN-beta and -alpha.

Domains of interaction between alpha interferon and its receptor components.

We describe how constraints on the binding of human interferons (IFNs), alpha1 and alpha2 and alpha8 on mouse cells are partially relieved by the expression of the bovine (Bo) or human (Hu) IFN alpha/beta receptor (IFNAR) component in these cells. We show that, while the binding of all three is substantially increased by the transfection of Bo IFNAR, it is accompanied by an increase in activity only in the case of alpha2 and alpha8 (IFNs that otherwise have little activity on mouse cells). IFN alpha1, which shows some partial activity on mouse cells, responds to the presence of Bo IFNAR by acting, at low concentrations, as a competitive antagonist to IFNs alpha2 and alpha8. A review of published results on IFN hybrid scanning and on the effects of expressing Bo IFNAR in human cells led us to propose that an N-terminal segment of the IFN molecule interacts directly with IFNAR. Applying site-directed mutagenesis to an IFN hybrid; alpha8[60]alpha1[92]alpha8, we show that the point mutations K84 to E84 and Y90 to D90 act synergistically to cause the hybrid to behave as the parental IFN alpha8, switching the preference from Mu to Hu IFNAR in transfected mouse cells. The published structural models for IFN reveal that positions 84 and 90 span the exposed residues of the alpha-helix C of the IFN molecule. We derive a model of IFN-receptor interaction in which the A helix and the C helix of IFN interact with IFNAR and in which a binding phase can be distinguished from a binding/activity phase. We propose that the so-called "hot" domains of the IFN molecule (the AB loop and the D helix) are presented by IFNAR to interact with an additional component of the functional receptor.

The interferon-induced Mx-homologous protein in people with symptomatic HIV-1 infection.

Twenty-six people with symptomatic HIV-1 infection were screened for the presence of interferon (IFN) alpha and IFN alpha antibodies in their sera and the presence of the IFN-induced intracellular Mx-homologous protein in their peripheral blood leukocytes. Eleven people had measurable IFN alpha levels ranging from 1 to 40 IU/ml. None of the sera tested was positive for IFN alpha binding or IFN alpha neutralizing antibodies in the assays employed. Twenty-five of the 26 people had significant levels of the Mx-homologous protein in their peripheral mononuclear cells. The Mx concentrations varied from 0.3 to 6 U/ml in the people studied. IFN alpha-positive people had significantly higher levels of the Mx homolog than IFN alpha-negative people (P less than 0.03). Furthermore, the Mx homolog content in Walter-Reed class 2 people was significantly lower than in Walter-Reed class 5/6 people (P less than 0.01). Our results suggest that the IFN system is activated in more than 90% of the people with lymphadenopathy-associated syndrome, AIDS-related complex and AIDS. Since acid-labile IFN alpha can induce the Mx homolog in vitro endogenously produced IFN alpha seems likely to be responsible for the high Mx homolog levels detected.

Correlation of the antiproliferative effect and the Mx-homologous protein induction by IFN in patients with malignant melanoma.

The human interferon-induced intracellular protein homologous to the murine Mx-protein has recently been identified by means of a specific monoclonal antibody. Three of six melanoma cell lines elicited this intracellular human Mx-homolog upon incubation with IFN-alpha or IFN-gamma, yet all six melanoma cell lines tested were susceptible to the antiproliferative effect of IFN-alpha and IFN-gamma. Compared per antiviral unit, IFN-gamma had weaker Mx-inducing but stronger antiproliferative activity than IFN-alpha. These data suggest that the IFN-induced Mx-homologous protein is not involved in the antiproliferative action of IFN on malignant melanoma cell lines. Furthermore, 51 patients with advanced malignant melanoma were treated thrice weekly with 10 x 10(6) IU rIFN-alpha-2b and 6 x 10(6) nIFN-alpha, respectively. Nine of the 51 patients experienced systemic objective tumor responses (3 complete response, 6 partial response), but had Mx concentrations in their mononuclear cells equal to the Mx levels of non-responders during IFN-alpha therapy. Therefore, the level of Mx-homologous protein induced during IFN therapy is not a predictive marker for an antitumor response in malignant melanoma.

Treatment strategies for early-stage chronic lymphocytic leukemia: can interferon-inducible MxA protein and tumor necrosis factor play a role as predictive markers for response to interferon therapy?

The potential benefit of interferon (IFN)-alpha therapy in early-stage B cell chronic lymphocytic leukemia (B-CLL) patients is still under discussion, and no assays are available to distinguish potential responders from nonresponders. Herein we analyzed the usefulness of serum tumor necrosis factor (TNF, a cytokine released by CLL cells) and MxA protein (an intracellular marker for biologic activity of endogenous IFN) concentrations as predictive measurements for evolution and response to IFN therapy in early-stage CLL patients. TNF levels and MxA expression were determined at diagnosis in 21 CLL patients. A statistically significant correlation was found between low TNF levels and MxA expression and between high TNF levels and no measurable MxA expression. The patients were then randomized to receive IFN-alpha or no therapy and were evaluated for response and evolution. When response to IFN-alpha therapy was considered, it became apparent that early-stage CLL patients with higher TNF levels and no measurable MxA expression were more likely to benefit from IFN therapy, whereas those patients with lower TNF levels and MxA expression could be considered CLL candidates for longer survival without therapy. More patients have to be tested to strengthen the value of MxA expression and TNF concentrations for subsequent response to IFN-alpha therapy.

Induction of Mx-related protein in cat peripheral blood mononuclear cells after administration of recombinant human interferon hybrid.

A 78 kDa protein was induced in cat peripheral blood mononuclear cells after in vivo administration of recombinant human interferon-alpha hybrid (rHuIFN-alpha B/D). This protein was antigenically related to the IFN-induced human (78 kDa) and mouse Mx proteins. Quantitative immunoblot analysis indicated that the induction of the cat Mx protein was dose-dependent. There was a dissociation in time between plasma levels of IFN which were transient, and levels of cat Mx protein which remained elevated at least five days after dosing. Our results provide evidence that a human IFN-alpha hybrid may be active in cats. They also indicate that the Mx protein is a sensitive, quantitative, and stable marker to follow IFN activity in vivo in cats.

Immunohistochemistry using antibodies to alpha-interferon and its induced protein, MxA, in Alzheimer's and Parkinson's disease brain tissues.

The localization of alpha-interferon (alpha-IFN) and its induced protein, MxA, was examined in human brain tissues from neurologically normal, Alzheimer's disease (AD) and Parkinson's disease (PD) cases. In all cases, a few neurons in the superficial cortical layers and microglial cells in the white matter were stained with the antibody to alpha-IFN. In AD brains, white matter microglia were intensely labeled for alpha-IFN and reactive microglia, such as those on senile plaques, were strongly positive for MxA protein. In PD, Lewy bodies in the substantia nigra were positive for MxA, but there was no staining for alpha-IFN in that region. These results suggest that increased expression of alpha-IFN in the white matter microglia and appearance of MxA protein in reactive microglia contribute to Alzheimer pathology. The staining of some Lewy bodies for MxA may be indicative of a viral infection or other unknown factor.

The action of recombinant bovine interferons on influenza virus replication correlates with the induction of two Mx-related proteins in bovine cells.

Recombinant bovine interferon-alpha and -gamma differ in their action against influenza virus on bovine cells. Bovine IFN-alpha severely impairs early protein synthesis and replication of influenza virus in bovine cells in contrast to bovine IFN-gamma which fails to induce an antiviral state against influenza virus. Otherwise the IFN system seems to function normally in bovine cells since both bovine IFN-alpha and -gamma induce an antiviral state against vesicular stomatitis virus. The establishment of the specific antiviral state against influenza virus correlates with the induction by bovine IFN-alpha, but not -gamma, of two cytoplasmic proteins related to the IFN-induced mouse protein Mx involved in the mechanism of resistance of mice to influenza virus infection. This study suggests that bovines possess a system for resistance to influenza virus similar to the mouse Mx system.

Virus-specific effects of recombinant porcine interferon-gamma and the induction of Mx proteins in pig cells.

Interferon-alpha (IFN-alpha) and -gamma differed in their action against influenza virus and vesicular stomatitis virus (VSV) on pig cells. Recombinant IFN-alpha severely impaired the cytopathic effect of VSV on PK-15 cells, whereas recombinant porcine IFN-gamma did not. IFN-alpha impaired also the replication of VSV and of influenza virus in primary pig kidney cells in contrast to IFN-gamma, which failed to induce an efficient antiviral state against both viruses. Otherwise, the IFN system seemed to work properly in pig cells since both IFN-alpha and IFN-gamma induced an efficient antiviral state to mengovirus. The establishment of the antiviral state to VSV and influenza virus correlated with the induction of two cytoplasmic proteins related to the murine Mx protein involved in the selective resistance of mice to influenza virus infection. The results are discussed in the context of the susceptibility of pigs to influenza virus strains that are in circulation in birds and in humans.

Interferons, Mx genes, and resistance to influenza virus.

Human influenza is primarily an infection of the upper respiratory tract and central airways. The interferon (IFN) system appears to have a role in limiting viral spread and initiating recovery before the development of T-cell and B-cell responses in primary infection. All cellular responses to IFNs result from interaction with cell surface receptors that trigger the expression of a number of cellular genes. Among the IFN-inducible gene products, the Mx proteins have attracted much attention because they have potential activity against influenza virus and possibly against other viruses. Mx proteins are guanosine triphosphate (GTP)-binding proteins with intrinsic GTPase activity. They seem to act indirectly against viruses by modifying cellular functions needed along the viral replication pathway. In mice the Mx1 protein has been shown to be necessary and sufficient to protect against influenza virus infection because the resistance does not require a functioning immune system. In humans the MxA protein has antiviral activities against influenza viruses. The MxA protein is encoded on the distal part of the long arm of chromosome 21 together with several other proteins implicated in the IFN system. Patients with Down's syndrome (trisomy 21) have an increased expression of MxA protein, and their cells display an increased sensitivity to IFNs in vitro because of gene dosage effects. These patients, however, are more susceptible to upper respiratory infection than normal individuals. This susceptibility has been related to deficiencies in the immune system. Therefore, induction of MxA in man does not sem sufficient to prevent influenza spreading, and, in contrast to the murine Mx system, a functioning immune system is necessary for protection.

Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins.

MxA is an abundant and ubiquitous cytoplasmic protein induced by alpha/beta interferon in human cells. Upon full induction, it can constitute 0.5 to 1% of cytosolic proteins. MxA can bind elements of the cytoskeleton, such as actin and tubulins, and several larger cellular proteins. However, these protein-protein interactions seem to be transitory. The human MxA protein contains a tripartite GTP-binding domain consisting of GxxxxGKS, DxxG, and TKxD, where x is any amino acid. It is shown here that the native MxA protein has GTPase activity (GTP----GDP) when purified by immunoprecipitation with affinity-purified polyclonal antibodies directed against the C-terminal domain of MxA. The GTPase activity is greatly diminished by polyclonal antibodies directed against the N-terminal domain of MxA (the domain which contains the GTP-binding consensus elements). Amino acid substitution within the GTP-binding domain abolished the GTPase activity of the mutated MxA protein expressed in transfected CHO cells. The reaction is specific for GTP, and the approximate Km is 0.1 mM. The reaction has an absolute requirement for Mg2+. The turnover number is approximately 70 molecules of GTP hydrolyzed per min per MxA molecule. It is suggested that the human MxA protein has certain characteristics of the stress proteins.

An interferon-induced mouse protein involved in the mechanism of resistance to influenza viruses. Its purification to homogeneity and characterization by polyclonal antibodies.

Interferon-alpha + beta (IFN-alpha + beta) plays a central role in the specific resistance to influenza virus infection of those mice carrying the gene Mx (for review, see Haller, O. (1981) Curr. Topics Microbiol. Immun. 92, 25). Particularly, mouse IFN-alpha + beta induces a unique protein in cultivated Mx-bearing cells which is associated with a highly efficient and specific antiviral resistance to influenza viruses (Horisberger, M. A., Staeheli, P., and Haller, O. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1910). In this report, a procedure is described for the induction of this protein in several organs of Mx-bearing mice and a method for its purification from liver tissue. The protein Mx is nucleophilic and has a Mr approaching 78,000. It is not concentrated in nucleoli and it is not tightly bound to chromatin or nuclear matrices. Polyclonal antibodies to the protein Mx were raised in BALB/c mice. They recognized the protein Mx immobilized on nitrocellulose in a dot immunoassay and they immunoprecipitated the IFN-induced protein Mx from cultivated Mx-bearing cells labeled with a radioactive tracer.

Combined action of mouse alpha and beta interferons in influenza virus-infected macrophages carrying the resistance gene Mx.

In mice, the combined action of alpha and beta interferons (IFNs) against influenza viruses is modulated by the host gene Mx. High concentrations of IFN fail to prevent efficiently the replication of influenza A virus in cultured macrophages lacking the gene Mx, whereas cultured macrophages carrying Mx develop strong antiviral activity even at low concentrations of IFN. Several steps in the replication cycle of influenza virus were compared in Mx/Mx and +/+ mouse macrophages treated with IFN-alpha + beta. Uncoating was not affected. A twofold reduction in the accumulation of primary transcripts was observed in IFN-treated macrophages at the highest concentration of IFN regardless of the genetic constitution of the host cell. No evidence was obtained for inhibition of influenza virus translation in macrophages which lacked Mx when treated with IFN-alpha + beta. In contrast, a marked shut-off of influenza virus polypeptide synthesis occurred in Mx-bearing macrophages treated with these IFNs, although the primary transcripts were active in directing the synthesis of viral polypeptides in a cell-free system. We concluded that a specific inhibitory mechanism for influenza virus translation was induced by IFN-alpha + beta in macrophages bearing the resistance gene Mx.

Interferon-induced proteins: identification of Mx proteins in various mammalian species.

Mx protein controls influenza virus pathogenicity in mice in vivo. It is an abundant protein synthesized in response to interferon-alpha/beta. Searches for homolog proteins in various animal species have been conducted using several methods: (1) radioactive labeling of proteins induced by interferon and analysis by 2-D gel electrophoresis, (2) immunoprecipitation, (3) protoblot-ELISA analysis of cell-protein extracts, and (4) immunostaining of fixed cells. All mammalian species tested so far (including human, horse, and pig, which are prone to influenza virus infection) responded to interferon-alpha by synthesizing one or two Mx proteins differing in relative molecular weight and pl. The expression of Mx proteins within one species differed sometimes between primary cultures of diploid cells and established cell lines. Mx proteins were detected in all species in the cytoplasm and, in some rodents only, also in the nucleus. This study indicates that the site of action of the Mx gene family might be cytoplasmic. The conservation and ubiquity of this family of proteins reflects a more general and essential cellular function than was initially considered.

Expression and stability of the Mx protein in different tissues of mice, in response to interferon inducers or to influenza virus infection.

We have defined some characteristics of the mouse Mx protein as a marker of biological response to interferon (IFN) and to virus infection in A2G mice. The Mx protein has been detected and quantitated by Western immunoblot analysis. Upon induction by poly(I):poly(C) or with Newcastle disease virus, the Mx protein is expressed and accumulated in a variety of organs, such as liver, lungs, spleen, kidneys, heart, and brain. In some organs the expression of the Mx protein is detected readily, as soon as 4 h after treatment. The highest protein level is reached at 24 h, and it remains stable for several days declining slowly to return to preinduced levels 2-3 weeks after treatment. Infection with an hepatotropic or a pneumotropic strain of influenza virus resulted in a systemic induction of Mx protein, the highest levels being found in the target organ for virus replication. Our results indicate that the Mx protein is a sensitive, quantitative, and stable marker to follow IFN activity or virus infection in an animal model.

IFN-alpha induced human 78 kD protein: purification and homologies with the mouse Mx protein, production of monoclonal antibodies, and potentiation effect of IFN-gamma.

A 78-kD protein (p78) is induced in human cells in response to interferon (IFN). It appeared as a radioactive spot when newly synthesized proteins from IFN-treated human cells labeled with [35S]methionine were separated in a two-dimensional system and autoradiographed. p78 was induced by IFN-alpha in normal human fibroblasts, and in some, but not all, established human tumor cell lines. It has been purified to homogeneity from Namalwa cells induced by recombinant IFN-alpha. Mouse polyclonal and monoclonal antibodies specific to p78 have been produced which allowed its quantitative determination in a Western blot ELISA. Using this method it was also shown that although IFN-gamma was a poor inducer of p78, it markedly increased the effect of IFN-alpha on p78 induction and accumulation. It was also demonstrated that p78 and the protein Mx of influenza-resistant mice, which we purified and characterized earlier (Horisberger and Hochkeppel, J. Biol. Chem. 260, 1730-1733, 1985) share common properties such as size, pI, amino acid composition, antigenic determinant(s), and IFN inducibility.

A recombinant human interferon-alpha B/D hybrid with a broad host-range.

A recombinant interferon (IFN) hybrid has been found to have a broad host-range of activity in an antiviral assay (plaque reduction of vesicular stomatitis virus) and also high efficacy as an antiviral agent in at least 12 different animal cell species. The IFN hybrid consists of amino acids 1 to 60 from HuIFN-alpha B and amino acids 61 to 166 from HuIFN-alpha D. The profile of cross-species activity of the IFN-alpha B/D hybrid has been compared with that of HuIFN-alpha F, and of the parents HuIFN-alpha B and -alpha D. When both IFN-alpha B and -alpha D were active in a cell species, the hybrid IFN had comparable or better activity than the more active parental IFN. The hybrid shared a broad cross-species activity with IFN-alpha D. However, the IFN-alpha B/D hybrid was 10-fold more active on human cells, 30-fold more active on rabbit cells, and 50-fold more active on mouse cells than IFN-alpha D.

Mx-dependent resistance to influenza viruses is induced by mouse interferons alpha and beta but not gamma.

In cells derived from congenic BALB.A2G-Mx mice carrying the resistance gene Mx, but not in cells from BALB/c mice lacking Mx, mouse interferons alpha and beta induced the synthesis of a unique cellular protein that was associated with an efficient antiviral state with selectivity for influenza viruses. In contrast, native or recombinant mouse interferon gamma failed to efficiently protect Mx-bearing cells against influenza viruses and did not noticeably induce the synthesis of the Mx-associated protein, although interferon gamma was as effective as interferons alpha and beta in protecting BALB.A2G-Mx and BALB/c cells against the rhabdovirus VSV. These results demonstrate that different types of interferons differentially regulate the expression of the Mx gene and thereby induce distinct antiviral states.