IFN-tau exhibits potent suppression of human papillomavirus E6/E7 oncoprotein expression.

The effects of interferon-tau (IFN-tau) on tumor suppressor factors and virus oncoprotein expression were compared with two other type I IFN in human papillomavirus (HPV-16)-transformed cells. Nontumorigenic human keratinocytes, HuKc/HPV-16d-2C (d-2C), treated with recombinant human IFN-alpha2a (Roferon), a human recombinant alpha IFN hybrid, alpha B/D (IFN-alphaB/D), or ovine IFN-tau were evaluated for their effects on the levels of E6 and E7 expression. IFN-tau was comparable to IFN-alpha2a in decreasing intracellular levels of E6 and E7, and IFN-alphaB/D was more effective than IFN-a2a in suppressing E7 levels. All three IFN were capable of increasing the cellular concentration of wild-type p53 tumor suppressor with the magnitude IFN-tau > IFN-alpha2a > IFN-alphaB/D. Increases in p53 concentrations correlated with the observed decreases in E6 mRNA and protein levels. The antiviral effects observed in this study reveal that IFN-tau has potent antipapillomavirus activity. Sequences/structures unique to IFN-tau could allow for alternative IFN/receptor interactions and may explain the differences in biologic function.

Molecular characterization of the hdm2-p53 interaction.

A number of viral oncogenes target the tumour suppressor protein p53 and inactivate its function. This is an important step in tumourogenesis. The cellular oncogene hdm2 acts through a similar mechanism. It binds the N terminus of p53, thereby interfering with the ability of p53 transcriptionally to activate genes responsible for growth arrest or apoptosis after genotoxic insults. The disruption of the interaction of the two proteins therefore comprises a promising therapeutic target for treatment of the subset of human cancers in which this pathway is active. In this paper we attempt to characterize the p53-hdm2 interaction biochemically. We analyse the potential of a series of peptide inhibitors, derived from previously described mdm2 binding peptide display phage, to disrupt this interaction in ELISA assays. We conclude that F19, W23 and L26 of p53 are critical contact points for p53 binding to hdm2. Furthermore, we show the potential of the monoclonal antibody 3G5 to interfere with binding of p53 to hdm2 in ELISA assays. Consequently, we define the binding site of 3G5 on hdm2 using overlapping peptides derived from the N terminus of hdm2 and phage display libraries. The result indicates L66, Y67 and E69 on hdm2 as critical binding points for 3G5. In electrophoretic mobility shift assay we demonstrate the formation of hdm2-p53 complexes that can be disrupted in the presence of 3G5 or inhibitory peptides. Finally, we describe the effects of NEM and DTT on the interaction between the two molecules in ELISA assays. All our results are discussed in the light of the recently published crystal structure of the mdm2-p53 complex. A striking correspondence between our findings and the crystal structure is revealed.

Identification of novel mdm2 binding peptides by phage display.

The oncogene mdm2 and its human homologue hdm2 bind to the tumour suppressor protein p53 and inactivate its function as a transcription factor. This has been implied as a possible mechanism for cancer development in several tumours including human sarcomas. The mdm2-p53 interaction is therefore a much persued target for the development of anti-cancer drugs. In order to find novel high affinity ligands for hdm2 which would interfere with its binding to p53 we screened phage display peptide libraries for mdm2 binding phage. We found a series of 12 and 15mer peptides which interact strongly with hdm2. The peptide sequences show striking homology with the previously established mdm2 binding site on p53, confirming that the peptide defined 18TFSDLW23 region is crucial for the interaction but that contact between the two molecules extends to position L26 on p53. Free synthetic peptides derived from the phage selected sequences proved to be up to 100 times stronger inhibitors of the p53-mdm2 interaction than the p53 derived wt-peptide in several ELISA-assays. This illustrates the potency of phage display libraries in the search for new peptide based lead structures designed to mimic or inhibit therapeutically important protein-protein interactions.

The effect of recombinant human interferon alpha B/D compared to interferon alpha 2b on SIV infection in rhesus macaques.

The model of simian immunodeficiency virus (SIV) infection in rhesus macaques was used to evaluate the effects of recombinant human interferon alpha, Hu IFN-alpha 2b and Hu IFN-gamma B,D, at two doses. Administration began 1 day prior to infection and was continued for 90 days postinfection. Both interferons suppressed SIV antigenemia during the treatment period. Following treatment animals were monitored for 4 years for rate of disease progression. Neither IFN prolonged the asymptomatic period or survival.

MxA gene expression after live virus vaccination: a sensitive marker for endogenous type I interferon.

MxA gene expression is known to be regulated tightly and exclusively by type I interferons (IFNs). The kinetics of MxA gene expression was analyzed in peripheral blood mononuclear cells from 11 healthy volunteers vaccinated with the 17-D strain of yellow fever virus. A reliable induction of MxA RNA and MxA protein was found in the absence of easily detectable serum IFN activity. Thus, steady-state MxA RNA levels were elevated 8- to 30-fold above prevaccination levels on day 5 after vaccination. The average increase of MxA protein was approximately 50-fold. In contrast, no induction of MxA RNA or MxA protein was detectable in 3 similarly vaccinated controls who were immune because of previous vaccinations. The IFN marker 2'-5'-oligoadenylate (2-5A) synthetase known to react to both type I and type II IFNs showed a similar response but did not differentiate equally well between nonimmune and immune vaccinees. beta 2-microglobulin and neopterin reacted poorly, remaining at low levels within the normal range. These results demonstrate that MxA gene expression is a good marker for detecting minute quantities of biologically active type I IFN during viral infections.

Strong transient expression of the type I interferon-induced MxA protein in hepatitis A but not in acute hepatitis B and C.

The human MxA protein is a new specific marker for type I interferon activity both in vitro and in vivo. In the study presented here, this interferon-induced marker, as well as the 2',5'-oligoadenylate synthetases, was measured in circulating mononuclear cells from 21 patients with acute hepatitis A, 20 patients with acute hepatitis B and 14 patients with acute hepatitis C for determination of the activation of the interferon system in these viral diseases. In acute hepatitis A a strong expression (10 of 10 patients) of the MxA protein and the 2',5'-oligoadenylate synthetase activity in peripheral-blood mononuclear cells was observed during the first 2 wk after onset of clinical symptoms. In this period the MxA protein concentrations reached levels similar to those measured in patients treated with up to 5 x 10(6) IU interferon-alpha three times a week. Beyond wk 3, in eight of eight patients with hepatitis A no increased MxA protein levels were found. In contrast, peripheral-blood mononuclear cells from patients with acute hepatitis B contained either no measurable MxA protein or only slightly higher levels of the MxA protein, as did those of most patients (12 of 14) with acute hepatitis C. The MxA protein levels of both hepatitis B and C patients were significantly lower (p < 0.05) than those found in hepatitis A patients. Furthermore, sera from 6 of 10 patients with hepatitis A, but none of 10 patients with acute hepatitis B and C, contained measurable MxA protein. This serum MxA protein may originate from interferon-exposed and subsequently damaged liver cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Effective natural interferon-alpha therapy in recombinant interferon-alpha-resistant patients with hairy cell leukemia.

To explore the relationship between anti-interferon-alpha (anti-IFN-alpha) antibodies and loss of clinical responsiveness to IFN-alpha treatment, we examined sera from 59 patients with hairy cell leukemia who responded to therapy with recombinant IFN-alpha-2a (rIFN-alpha-2a). During the first 2 years of therapy, 10 patients developed rIFN-alpha-2a-neutralizing and 15 rIFN-alpha-2a-binding antibodies. Nine of the 59 initially responding patients became resistant to rIFN-alpha-2a and suffered a relapse of the disease at 7 to 24 months of treatment. All nine relapsing patients tested positive for both neutralizing and binding antibodies with titers above 400 INU/mL, while none of the antibody-negative patients relapsed. Six patients with detectable binding antibody titers below 400 INU/mL continued to respond to treatment. By measuring the IFN kinetics and the levels of the IFN-induced Mx-homologous protein in mononuclear cells after a single injection each of rIFN-alpha-2a and nIFN-alpha the IFN antibodies of eight of the nine resistant rIFN-alpha patients were found to be highly specific for rIFN-alpha-2a. Therefore, these eight patients were switched to natural IFN-alpha (nIFN-alpha) therapy at doses of 3 million IU, three times a week. All eight patients responded to treatment with nIFN-alpha, achieving durable objective responses similar to those obtained previously with rIFN-alpha-2a. These data clearly demonstrate that rIFN-alpha antibody-positive patients can effectively be treated with nIFN-alpha.

Treatment of anti-recombinant interferon-alpha 2 antibody positive CML patients with natural interferon-alpha.

Of 38 patients with a Philadelphia-chromosome positive chronic myeloid leukaemia treated with recombinant interferon alpha (rIFN-alpha) 2a or 2b and monitored for emergence of IFN-antibodies in their sera 11 patients developed rIFN-alpha 2 binding and 10 rIFN-alpha 2 neutralizing antibodies. rIFN-alpha neutralizing antibody positive patients experienced significantly (P less than 0.025) more clinical relapses (6/10) than IFN-antibody negative patients (6/28) during continuous IFN-therapy. Furthermore, IFN-antibody-positive patients with titre above 400 INU/ml were more likely to relapse under rIFN-alpha-therapy than IFN-antibody-negative patients with titre below 400 INU/ml (P less than 0.05). Seven rIFN-antibody-positive patients experiencing a clinical relapse or a primary non-responsiveness were treated with two- to three-fold increased doses of rIFN-alpha 2. Only one of these seven patients developed a partial haematological remission upon intensification of the rIFN-alpha 2 therapy. Consecutively, the six patients failing high dose rIFN-alpha treatment were switched to a natural IFN-alpha preparation (3 x 9 x 10(6) I.U. weekly s.c.). Under such treatment two of the six patients achieved a long-lasting complete, one a partial haematological remission. In high-titred IFN-antibody positive patients significantly altered serum-IFN-titre and minimal IFN-inducible Mx-homologue concentrations were measured; in contrast, nIFN-alpha induced normal IFN-titre and dose-equivalent Mx-homologue amounts in these patients. The data prove that high-titred rIFN-alpha neutralizing antibodies abrogate the biological action of rIFN-alpha, but not of nIFN-alpha in vivo and explains why nIFN-alpha can be effective in the anti rIFN-alpha 2 positive patients.

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.

The human intracellular Mx-homologous protein is specifically induced by type I interferons.

The murine Mx-1 protein is one of the best biochemically and functionally characterized interferon (IFN)-induced proteins that is necessary, and sufficient, for providing resistance to murine cells against viral influenza infection. Recently an intracellular human protein homologous to the murine Mx-1 protein has been identified by means of a specific monoclonal antibody. The restricted induction of this intracellular protein in human mononuclear cells (MNC) by various cytokines was investigated. MNC from 26 of 28 healthy people and 35 of 36 cancer patients before IFN-alpha therapy had no detectable Mx-homologous protein. Incubation of human MNC with IFN-alpha and IFN-beta for 24 h at different concentrations led to a dose-dependent induction of the Mx-homologous protein. All IFN-alpha or IFN-beta preparations tested were equally effective in eliciting this intracellular protein. IFN-gamma induced only 1% of the Mx amount elicited by type-1 IFN compared on a weight basis. Neither interleukin (IL) 1 nor IL3, IL4, IL5, IL6, tumor necrosis factor-alpha/beta, granulocyte colony-stimulating factor (CSF) or granulocyte macrophage-CSF at any of the concentrations tested were capable of eliciting any detectable amount of the Mx homolog, while IL2 was a poor Mx-homologous protein inducer. In the presence of high-titered IFN-alpha antisera both IL2 and IFN-gamma were unable to stimulate this protein, proving that IFN-gamma and IL2 indirectly induce the Mx homolog via IFN-alpha. Therefore, the human Mx-homologous protein is a strictly by type I IFN-regulated protein in human peripheral blood lymphocytes.

Emergence and decay of the human Mx homolog in cancer patients during and after interferon-alpha therapy.

The human Mx, an interferon (IFN)-alpha- and IFN-beta-induced 76-kd protein, is a homolog (Mx-homolog) to the murine Mx protein, which is necessary and sufficient to provide adequate resistance against influenza virus in murine cells and in mice. Leukocytes from 36 patients with tumors (chronic myelogenic leukemia, hairy cell leukemia, and malignant melanoma) were monitored for their Mx-homolog content before, during, and after rIFN-alpha-2b therapy. Before therapy, only one patient was slightly positive for Mx-homolog. All 36 patients showed a significant increase of Mx-homolog in their mononuclear cells within the first day of IFN therapy. During therapy, the Mx-homolog levels remained elevated. After cessation of treatment, the Mx-homolog content in the mononuclear cells decreased slowly; within 2 weeks, it was about 20-30% of its value during therapy. However, even after 3 weeks, the Mx-homolog was still detectable. The maximally induced Mx-homolog concentration showed a significant correlation to the IFN dose given in vivo. These data indicate that the Mx-homolog is an excellent marker for monitoring the activity of IFN during IFN therapy. In addition, the in vivo endogenous activation of the IFN system might be detectable by the determination of the Mx-homolog despite the lack of circulating IFN.

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.

9-(2-Phosphonylmethoxyethyl)adenine in the treatment of murine acquired immunodeficiency disease and opportunistic herpes simplex virus infections.

The murine model of acquired immunodeficiency disease was used to evaluate both the antiretroviral and antiherpetic activities of the acyclic nucleotide analog 9-(2-phosphonylmethoxyethyl)adenine (PMEA). The antiretroviral activity of PMEA was compared with that of azidothymidine (AZT) in mice receiving the drug either immediately after infection or at late times in disease progression. Both AZT (oral, 30 mg/kg) and PMEA (parenteral, 25 and 5 mg/kg) were effective in slowing the development of disease when administered daily beginning on the day of infection. In contrast, neither drug alone was effective in modifying disease outcome when administered several weeks after viral infection. Human recombinant alpha interferon (rhuIFN alpha-B/D at 5 x 10(7) U/kg) was also ineffective when administered late in the course of disease. However, when administered in combination, both alpha interferon and PMEA (25 mg/kg) were able to suppress disease progression even when treatment was initiated as late as 3 weeks postinfection. Mice that were immunocompromised due to LP-BM5 virus infection were highly susceptible to acute (lethal) infection with herpes simplex virus type 1, whereas their immunocompetent littermates were not. PMEA was as effective as acyclovir in the treatment of opportunistic herpes simplex virus type 1 infections in LP-BM5 virus-infected mice. Thus, like AZT, PMEA was effective against retrovirus infection, and, like acyclovir, PMEA was effective against herpes simplex virus type 1 infection. This gives PMEA the unique potential of being useful in the treatment of opportunistic herpes simplex virus infections as well as the underlying retroviral disease.

MX homologous protein in mononuclear cells from patients with systemic lupus erythematosus.

The 76-kd human interferon (IFN)-induced MX protein is the homolog to the murine protein, which is necessary and sufficient to provide adequate resistance to influenza virus infection in murine cells and in mice. Fifty-one patients with systemic lupus erythematosus (SLE) were screened for the presence of the MX homolog in mononuclear cells and for IFN and anti-IFN antibodies in serum. In 47 of 51 patients, significant levels of the MX homolog were found, while only 15 of 51 patients had measurable alpha-IFN in their serum. The IFN activity found in these sera was characterized as a partially acid-labile alpha-IFN, by means of acid-stability cross-reactivity on heterologous cells, trypsin sensitivity, and neutralization by homologous or heterologous antisera. Four of the patients had no detectable MX homolog in their leukocytes; 3 of these 4 possessed an anti-alpha-IFN antibody that was able to neutralize both a natural alpha-IFN preparation and the acid-labile IFN in SLE sera. Also, acid-labile alpha-IFN-containing SLE sera induced the MX homolog in vitro in mononuclear cells from healthy donors. These observations suggest that endogenously produced alpha-IFN is responsible for the observed induction of the MX homolog in SLE and that the IFN system is activated in more than 90% of SLE patients.

Significant differences in therapeutic responses to a human interferon-alpha B/D hybrid in Rauscher or Friend murine leukemia virus infections.

Treatment of mice infected with Rauscher (RMLV) or Friend (FMLV) murine leukemia viruses at an early stage of disease (beginning at day 0 and continuing every other day for 21 days) with 5 x 10(7) units/kg body weight of a cross-species-active recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D) was more effective in FMLV than in RMLV infections. In contrast, treatment with 5 x 10(7) units/kg body weight of IFN beginning as late as 15 days postinfection and continuing every other day for 21 days was more effective in RMLV than in FMLV infections. These differences were consistent with observed changes in circulating white blood cells, spleen weight, and reverse transcriptase levels. Additionally, biweekly long-term administration (beginning at day 0) of 5 x 10(6) units/kg of rHuIFN-alpha B/D (an ineffective treatment in short-term therapy) significantly prolonged the mean survival time of RMLV-infected mice, but only weakly prolonged that of FMLV-infected mice.

The immunomodulatory effects of interferon-gamma on mature B-lymphocyte responses.

Interferon-gamma (IFN-gamma) exerts a broad spectrum of activities which affect the responses of mature B-cells. It strongly inhibits B-cell activation, acts as a B-cell growth factor (BCGF), and also induces final differentiation to immunoglobulin (Ig) production. IFN-gamma is deeply involved in the differential control of isotype expression, as it enhances IgG2a production and suppresses both IgG1 and IgE production. Although it is now possible to draw a general scheme of the effects of IFN-gamma on B-cells, a number of paradoxical results still exist in the field. In this manuscript, different experimental systems are analyzed in an attempt to explain these apparent paradoxes.

Antiviral activity of a novel recombinant human interferon-alpha B/D hybrid.

The antiviral potential of a novel cross-species active, recombinant human interferon-alpha B/D hybrid (rHuIFN-alpha B/D), was evaluated for its efficiacy in cultured human monocytes and in several murine models of viral disease. When examined in 14-day-old human monocyte cultures, rHuIFN-alpha B/D was highly effective in preventing viral replication and cell destruction caused by herpes simplex virus type 1 (HSV-1/VR3). The effect observed with 100 units of this hybrid IFN was as good or higher than that observed with equivalent amounts of rHuIFN-alpha A or IFN-gamma. In addition, a single dose (5 X 10(7) U/kg) of rHuIFN-alpha B/D administered several hours after intranasal infection with HSV-1/VR3 suppressed pulmonary virus replication and prevented death due to interstitial pneumonia. Similarly, mice infected with a more aggressive strain of HSV-1 (McIntyre) were protected when this IFN preparation was administered at the time of virus infection and 1 day later. The anti-retroviral activity of rHuIFN-alpha B/D was examined in two murine leukemia retroviral models, Rauscher (RMLV) and Friend (FMLV), and a murine model of acquired immunodeficiency (LP-BM5). Treatment of RMLV or FMLV infected mice significantly prolonged mean survival times and the number of long-term FMLV survivors. These therapeutic effects were demonstrated when IFN was administered on the day of virus infection or as late as 3 days following infection. Transient reversal of the immunosuppressive effects induced by LP-BM5 infection was observed when rHuIFN-alpha B/D treatment was initiated at the time of virus infection. Moreover, when rHuIFN-alpha B/D was used together with azidothymidine (AZT), the effect of the combination was better than either drug alone.

Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis.

Chronic respiratory tract infections caused by Staphylococcus aureus are common in patients with cystic fibrosis (CF). Recently, it was shown in a few CF patients that S. aureus isolates produce capsular polysaccharides (CPs). However, it is not known whether this is a common feature and whether an immune response to CPs in CF is detectable. Therefore, we examined 170 S. aureus isolates from CF patients and healthy individuals for production of CP types 5 and 8 by using monoclonal antibodies. We found that CP-producing staphylococcal isolates were randomly distributed among CF patients and healthy carriers. Eighty-five percent of all isolates produced CPs, 77% of which were type 8. Examination of one sputum sample by an immunofluorescence technique suggested that production of CPs is not an in vitro phenomenon. S. aureus isolates from various sites of a single person often yielded more than one CP type. A random distribution of S. aureus strains with CP type 5 or 8 from the skin and respiratory tracts of patients and from the skin of healthy individuals was found. Antibody response to CP types 5 and 8, measured by enzyme-linked immunosorbent assay, was not elevated in CF patients with chronic S. aureus lung infection in comparison with healthy carriers. On the contrary, in CF patients the ratios of antibodies to CP 8 were significantly lower (P less than 0.005; alpha = 0.025). The ratios of antibodies to CP types did not change when monitored longitudinally over several months. This study suggests that the production of CPs is a universal property of S. aureus and that infected CF patients do not have elevated ratios of antibodies to these antigens.

Enhanced antiproliferative action of interferon targeted by bispecific monoclonal antibodies.

It has previously been shown that interferon (IFN) can be coupled covalently to tumor-specific monoclonal antibodies (mAb) and that the in vitro antiviral and antiproliferative action of these IFN-mAb conjugates is superior to that of uncoupled IFN. We now report a different mode of IFN delivery, i.e., via bispecific mAbs, avoiding chemical coupling of IFN. Bispecific mAbs were prepared by cross-linking two mAbs with SPDP, mAb1 being specific for an idiotype of a hybridoma cell-surface immunoglobulin and mAb2 specific for an IFN. Alternatively, Fab' fractions of mAb1 and mAb2 were coupled by disulfide formation to produce F(ab')2. Binding capacity and specificity of both arms of the mAb conjugates were first demonstrated by a solid-phase radioimmunoassay using idiotype-positive mAb as test antigen and 125I-labeled hybrid IFN-alpha B/D. Secondly, hybridomas either idiotype positive or negative were incubated with bispecific mAbs (mAb1-mAb2 or Fab'1-Fab'2) and 125I-labeled IFN at 4 degrees C. After washing away unbound reagents, the uptake of radioactivity into cells was determined. Additionally, the antiproliferative action of cold or labeled IFN targeted via different modes was assessed by an [3H]TdR incorporation method. Results showed that bispecific mAbs could specifically deliver IFN to the target cells and also inhibit their growth in vitro. Furthermore, targeting IFN by any of the three methods, IFN-mAb, mAb1-mAb2, or Fab'1-Fab'2, enhanced its in vitro antiproliferative potency compared to IFN alone.