An Albumin-Free Formulation for Escherichia coli-Derived Interferon Beta-1b with Decreased Immunogenicity in Immune Tolerant Mice.

Human serum albumin (HSA)-free formulation of Escherichia coli-derived human interferon beta (IFNß-1b) with a high percentage of monomeric protein and low immunogenicity is developed and characterized in the current study. UV spectroscopy, fluorescence spectroscopy, dynamic light scattering, sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, Micro-Flow Imaging, resonant mass measurement, size exclusion, and reversed-phase high performance liquid chromatographies were applied to assess the effect of excipients on the stability of IFNß-1b to establish a HSA-free formulation. The antiviral activity of IFNß-1b was evaluated using human lung carcinoma cell line. Immune tolerant mice to hIFNß were used to assess the immunogenicity of the HSA-free formulated IFNß-1b in comparison to Betaferon drug product and Avonex drug substance as standards through IgG titering of plasma. HSA-free formulated IFNß-1b, including 200 mM L-arginine, 200 mM trehalose, and 0.1% n-dodecyl ß-D-maltoside in 10 mM sodium acetate buffer, pH 7.4, showed the highest biological activity. The stability of IFNß-1b in the HSA-free formulation was monitored for 3 weeks at 4°C and 37°C with relative humidity of 10% and 75%, respectively. Protein aggregation and immunogenicity in transgenic mice were decreased in the HSA-free formulated IFNß-1b compared to Betaferon. The stability, biological activity, and immunogenicity of the HSA-free formulation and Betaferon were evaluated. Incubation of formulations at 4°C and 37°C for 3 weeks showed that the HSA-free formulated IFNß-1b was more stable and less immunogenic in transgenic FVB/N mice. Low immunogenicity and the absence of HSA, which reduces the potential risk of viral infection (eg, HIV and HCV), are promising for clinical studies.

Small Molecule Agonists for the Type I Interferon Receptor: An In Silico Approach.

Type I interferons (IFNs) exhibit broad-spectrum antiviral activity, with potential utility against emerging acute virus infections that pose a threat to global health. Recombinant IFN-αs that have been approved for clinical use require cold storage and are administered through intramuscular or subcutaneous injection, features that are problematic for global distribution, storage, and administration. Cognizant that the biological potency of an IFN-α subtype is determined by its binding affinity to the type I IFN receptor, IFNAR, we identified a panel of small molecule nonpeptide compounds using an in silico screening strategy that incorporated specific structural features of amino acids in the receptor-binding domains of the most potent IFN-α, IFN alfacon-1. Hit compounds were selected based on ease of synthesis and formulation properties. In preliminary biological assays, we provide evidence that these compounds exhibit antiviral activity. This proof-of-concept study validates the strategy of in silico design and development for IFN mimetics.

Total chemical synthesis of human interferon alpha-2b via native chemical ligation.

Interferon-alpha (IFNα) is a cytokine that orchestrates innate and adaptive immune responses and potently inhibits proliferation of normal and tumor cells. These properties have warranted the use of IFNα in clinical practice for the treatment of several viral infections and malignancies. However, overexpression of IFNα leads to immunopathology observed in the context of chronic viral infections and autoimmune conditions. Thus, it is desirable to develop therapeutic approaches that aim at suppressing excessive IFNα production. To that end, artificial evolution of peptides from phage display libraries represents a strategy that seeks to disrupt the interaction between IFNα and its cell surface receptor and thus inhibit the ensuing biological effects. Mirror-image phage display that screens peptide libraries against the D-enantiomer is particularly attractive because it allows for identification of proteolysis-resistant D-peptide inhibitors. This approach, however, relies on the availability of chemically synthesized D-IFNα composed entirely of D-amino acids. Here, we describe the synthesis and biological properties of IFNα2b of 165 amino acid residues produced by native chemical ligation, which represents an important first step toward the discovery of D-peptide antagonists with potential therapeutic applications.

Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor-2 inhibits type 1 interferon signalling by targeting interferon-stimulated gene factor-3.

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes four viral interferon regulatory factors (vIRF-1-4). We investigated the mechanism and consequences of vIRF-2-mediated inhibition of interferon-response element signalling following type I interferon (IFN) induction. Western blot and electrophoretic mobility-shift assays identified the interferon-stimulated gene factor-3 (ISGF-3) components STAT1 and IRF-9 as the proximal targets of vIRF-2 activity. The biological significance of vIRF-2 inhibition of ISGF-3 was demonstrated by vIRF-2-mediated rescue of the replication of the IFN-sensitive virus encephalomyocarditis virus. This study provides both a mechanism and evidence for KSHV vIRF-2-mediated suppression of the consequences of type 1 IFN-induced signalling.

Genetic redundancy in human cervical carcinoma cells: identification of cells with "normal" properties.

Although it is generally assumed that cancer arises from a singular cell, a tumor must be considered as a dynamic and emergent biological structure, whose organizing principle is determined by genetic and epigenetic modifications, occurring variably in response to microenvironmental selection conditions. As previously shown, HPV-positive cervical carcinoma cells have lost their ability to induce IFN-beta upon TNF-alpha treatment. However, regarding cancer as a non-linear system, which may, even in the absence of an apparent selection pressure, fluctuate between different "metastable" phenotypes, we demonstrate that TNF-alpha mediated IFN-beta induction is not irreversibly disturbed in all cells. Using the IFN-beta sensitive Encephalomyocarditis virus (EMCV) as a tool to monitor antiviral activity in long-term established malignant HeLa cells, rare IFN-beta expressing clones were rescued from a population of non-responsive and EMCV-sensitive cells. Antiviral activity was mediated by the re-expression of IRF-1 and p48 (IRF-9), both key regulatory molecules normally found to be suppressed in cervical carcinoma cells. Upon inoculating of selected clones into immunocompromised animals, a reduced or even an absence of tumorigenicity of initially highly malignant cells could be discerned. These data indicate that both the absence of interferon signaling and the ability to form tumors were reversed in a minority of cells. We provide a paradigm for the existence of innate genetic redundancy mechanisms, where a particular phenotype persists and can be isolated without application of drugs generally changing the epigenetic context.

Effects of tacrolimus (FK506) on encephalomyocarditic virus-induced diabetes in mice.

The effects of tacrolimus on insulin-dependent diabetes mellitus (IDDM) induced by the D-variant of encephalomyocarditis virus (D-EMCV) have been investigated. Male BALB/c mice were treated with tacrolimus before viral inoculation, and then were inoculated with 10 plaque forming units (PFU) of DEMCV. The mice continued to be treated with tacrolimus until the animals were sacrificed. D-EMCV-infected mice, which were treated with saline as controls, showed abnormal glucose tolerance test (GTT) values, whereas all infected mice with tacrolimus pretreatment were normal on 7 days-post inoculation (DPI). Histological observations revealed that non-treated tacrolimus D-EMCV-infected mice and which developed diabetes showed severe insulitis in their islets of Langerhans. On the other hand, D-EMCV-infected mice treated with tacrolimus were normal. In D-EMCV-infected mice, viruses in the pancreata were detected at the same level regardless of treatment with tacrolimus or saline. Expressions of TNF-alpha and IFN-gamma mRNA in spleens of tacrolimus-treated D-EMCV-infected mice were lower than that of non-treated tacrolimus DEMCV-infected mice on 7 DPI. The results suggest that tacrolimus suppresses expressions of TNF-alpha and IFN-gamma mRNAs to prevent the onset of D-EMCV-induced IDDM.

Improved pharmacokinetic properties of a polyethylene glycol-modified form of interferon-beta-1a with preserved in vitro bioactivity.

Interferon therapies suffer from a relatively short half-life of the products in circulation. To address this issue we investigated the effects of polyethylene glycol modification (PEGylation) on the pharmacokinetic properties of human interferon (IFN)-beta-1a. PEGylation with a linear 20-kDa PEG targeted at a single site on the N-terminal amine had no deleterious effect on its specific activity in an in vitro antiviral assay. In monkeys, PEG IFN-beta-1a treatment induced neopterin and beta2-microglobulin expression (pharmacodynamic markers of activity). Systemic clearance values in monkeys, rats, and mice decreased, respectively, from 232, 261, and 247 ml/h/kg for the unmodified IFN-beta-1a to 30.5, 19.2, and 18.7 ml/h/kg for the PEGylated form, while volume of distribution values decreased from 427, 280, and 328 ml/kg to 284, 173, and 150 ml/kg. The decreased clearance and volume of distribution resulted in higher serum antiviral activity in the PEG IFN-beta-1a-treated animals. In the rat, a more extensive set of dosing routes was investigated, including intraperitoneal, intratracheal, and oral administration. Bioavailability for the PEG IFN-beta-1a was similar to the unmodified protein for each of the extravascular routes examined. For the intraperitoneal route, bioavailability was almost 100%, whereas for the oral and intratracheal routes absorption was low (<5%). In rats, subcutaneous bioavailability was moderate (28%), whereas in monkeys it was approximately 100%. In all instances an improved pharmacokinetic profile for the PEGylated IFN-beta-1a was observed. These findings demonstrate that PEGylation greatly alters the pharmacokinetic properties of IFN-beta-1a, resulting in an increase in systemic exposure following diverse routes of administration.

Study on risk factors for transplacental viral infections; effect of bacterial factors and double viral infections on virus replication in placenta and amniotic membranes.

Among risk factors for vertical transmission of HIV there are listed concomitant viral and bacterial infections. Therefore the influence on the viruses replication in human placenta and amniotic membrane cultures of double viral infection with two unrelated viruses - encephalomyocarditis (EMCV) and vesicular stomatitis virus (VSV) - was studied and compared with the replication of the viruses in single virus infection (EMCV or VSV) in the same organ cultures. Additionally effect of bacterial factors - lipopolysaccharide (LPS) Escherichia coli and sonicated Treponema pallidum antigens (Tpa) - on VSV replication in the same culture system was studied and compared with VSV replication in untreated explants. Two effects were observed in double-virus infected cultures and also in bacterial factors treated cultures: inhibition and stimulation of virus replication. The kind of effect in the both cases was dependent on the presence or absence of innate antiviral immunity. In virus-sensitive organs double infected or treated with LPS or Tpa, inhibition of virus titer (2-5 log TCID(50)/ml) was observed. In the organs expressing the innate immunity, stimulation (1-4 log TCID(50)/ml) of virus replication was noticed. Contribution of endogenous TNFalpha in both reactions (stimulation and inhibition) was confirmed using antibodies against the TNF.

Leader protein of encephalomyocarditis virus binds zinc, is phosphorylated during viral infection, and affects the efficiency of genome translation.

Encephalomyocarditis virus (EMCV) is the prototype member of the cardiovirus genus of picornaviruses. For cardioviruses and the related aphthoviruses, the first protein segment translated from the plus-strand RNA genome is the Leader protein. The aphthovirus Leader (173-201 amino acids) is an autocatalytic papain-like protease that cleaves translation factor eIF-4G to shut off cap-dependent host protein synthesis during infection. The less characterized cardioviral Leader is a shorter protein (67-76 amino acids) and does not contain recognizable proteolytic motifs. Instead, these Leaders have sequences consistent with N-terminal zinc-binding motifs, centrally located tyrosine kinase phosphorylation sites, and C-terminal, acid-rich domains. Deletion mutations, removing the zinc motif, the acid domain, or both domains, were engineered into EMCV cDNAs. In all cases, the mutations gave rise to viable viruses, but the plaque phenotypes in HeLa cells were significantly smaller than for wild-type virus. RNA transcripts containing the Leader deletions had reduced capacity to direct protein synthesis in cell-free extracts and the products with deletions in the acid-rich domains were less effective substrates at the L/P1 site, for viral proteinase 3Cpro. Recombinant EMCV Leader (rL) was expressed in bacteria and purified to homogeneity. This protein bound zinc stoichiometrically, whereas protein with a deletion in the zinc motif was inactive. Polyclonal mouse sera, raised against rL, immunoprecipitated Leader-containing precursors from infected HeLa cell extracts, but did not detect significant pools of the mature Leader. However, additional reactions with antiphosphotyrosine antibodies show that the mature Leader, but not its precursors, is phosphorylated during viral infection. The data suggest the natural Leader may play a role in regulation of viral genome translation, perhaps through a triggering phosphorylation event.

Mengovirus and encephalomyocarditis virus poly(C) tract lengths can affect virus growth in murine cell culture.

Many virulent aphthoviruses and cardioviruses have long homopolymeric poly(C) tracts in the 5' untranslated regions of their RNA genomes. A panel of genetically engineered mengo-type cardioviruses has been described which contain a variety of different poly(C) tract lengths. Studies of these viruses have shown the poly(C) tract to be dispensable for growth in HeLa cells, although the relative murine virulence of the viruses correlates directly and positively with tract length. Compared with wild-type mengovirus strain M, mutants with shortened poly(C) tracts grow poorly in mice and protectively immunize rather than kill recipient animals. In the present study, several murine cell populations were tested to determine whether, unlike HeLa cells, they allowed a differential amplification of viruses with long or short poly(C) tracts. Replication and cytopathic studies with four hematopoietically derived cell lines (CH2B, RAW 264.7, A20.J, and P815) and two murine fibroblast cell lines [L929 and L(Y)] demonstrated that several of these cell types indeed allowed differential virus replication as a function of viral poly(C) tract length. Among the most discerning of these cells, RAW 264.7 macrophages supported vigorous lytic growth of a long-tract virus, vMwt (C(44)UC(10)), but supported only substantially diminished and virtually nonlytic growth of vMC(24) (C(13)UC(10)) and vMC(0) short-tract viruses. The viral growth differences evident in all cell lines were apparent early and continuously during every cycle of virus amplification. The data suggest that poly(C) tract-dependent attenuation of mengovirus may be due in part to a viral replication defect manifest in similar hematopoietic-type cells shortly after murine infection. The characterized cultures should provide excellent tools for molecular study of poly(C) tract-mediated virulence.

Induction of the human protein P56 by interferon, double-stranded RNA, or virus infection.

P56 is the most abundant protein induced by interferon (IFN) treatment of human cells. To facilitate studies on its induction pattern and cellular functions, we expressed recombinant P56 as a hexahistidine-tagged protein in Escherichia coli and purified it to apparent homogeneity using affinity chromatography. A polyclonal antibody raised against this recombinant protein was used to show that P56 is primarily a cytoplasmic protein. Cellular expression of P56 by transfection did not inhibit the replication of vesicular stomatitis virus and encephalomyocarditis virus. P56 synthesis was rapidly induced by IFN-beta, and the protein had a half-life of 6 h. IFN-gamma or poly(A)(+) could not induce the protein, but poly(I)-poly(C) or an 85-bp synthetic double-stranded RNA efficiently induced it. Similarly, infection of GRE cells, which are devoid of type I IFN genes, by vesicular stomatitis virus, encephalomyocarditis virus, or Sendai virus caused P56 induction. Surprisingly, Sendai virus could also induce P56 in the mutant cell line P2.1, which cannot respond to either IFN-alpha/beta or double-stranded RNA. Induction of P56 in the P2.1 cells and the parental U4C cells by virus infection was preceded by activation of IRF-3 as judged by its translocation to the nucleus from the cytoplasm.

Heat stress aggravates viral myocarditis in mice.

While a beneficial effect of hyperthermia on viral infection has been hypothesized, there are no data on viral myocarditis in vivo. To investigate whether hyperthermia might attenuate the course or severity of viral myocarditis, we studied the pathological changes in a murine model of viral myocarditis. C3H mice were inoculated i.p. with the encephalomyocarditis virus (500 pfu). They were anesthetized and heated to a body temperature of 42.5+/-0.2 degrees C for 30 min. The latter was performed 4 hr before (n=28, HB) or 4 hr after (n=28, HA) the viral inoculation; results were compared with nonheated, infected controls (n=30, Cont). Cardiac viral titers were recorded on day 3, and the body weight (BW), heart weight (HW) and pathological changes were recorded on days 5 and 10. The incidence of spontaneous mortality on day 10 was significantly higher in the HA group (all deaths occurring by day 7 post-inoculation) as compared with the HB (35%) or Cont (18%) groups. Viral titers in the HA group (n=4) were significantly (P<0.05) higher than those in the Cont (n=7) or HB (n=7) groups (4.11+/-0.54 vs 3.01+/-0.44 and 3.23+/-0.45 LogTCID50/mg, respectively). On day 5, the HW, the BW/HW ratio, and the severity of myocardial necrosis were all significantly higher in the HA than in the Cont and HB groups. To confirm the effect of hyperthermia on the expression of heart shock protein (HSP), immunohistochemical staining was done in the virus-infected hearts. The nucleus and cytoplasm of the injured myocardium in the HA group strongly expressed HSP70, whereas the HB and Cont groups were negative for this protein. In conclusion, induction of hyperthermia after viral inoculation aggravated the viral-induced myocardial necrosis and increased the mortality rate in a murine model of viral myocarditis and induced myocardial heat shock protein 70.

Pharmacological biocompatibility between intravesical preparations of BCG and interferon-alpha 2B.

PURPOSE: To determine if BCG and interferon alpha-2B are mutually compatible as mixed intravesical agents for clinical bladder cancer therapy. MATERIALS AND METHODS: Mutual compatibility was assessed by measuring IFN-alpha's effect on BCG metabolic activity, growth rate, and clumping tendency and conversely by observing BCG's effect on IFN-alpha's anti-viral activity. Optical density at 600 nm. (OD600) was used to estimate the number of colony forming units of BCG in suspension during 3 hours measurements of clumping and 8 days measurements of BCG proliferation. BCG viability was evaluated using a substrate marker, MTT, which correlates with BCG density and metabolic activity. The anti-viral activity of IFN-alpha was determined in a cytopathic protection bioassay using the encephalomyocarditis virus/FS-4 cell system. RESULTS: Continuous shaking of reconstituted BCG for 3 hours at 37C resulted in a marginal (11.3%) drop in OD600 which was minimally altered by inclusion of IFN-alpha at 2 million units (MU)/ml. (12.7% drop). Metabolic activity and growth rate of BCG alone or BCG with IFN-alpha were essentially identical. IFN-alpha's antiviral activity was not affected by incubation with BCG. CONCLUSIONS: The inclusion of IFN-alpha into the usual BCG formulation for intravesical administration has no apparent effect on BCG's viability or tendency to form clumps in suspension. Similarly, the physical mixing of IFN-alpha with BCG does not impair its biological activity. Thus, both agents are pharmacologically compatible for future clinical studies involving combination intravesical therapy.

Involvement of the double-stranded-RNA-dependent kinase PKR in interferon expression and interferon-mediated antiviral activity.

The signaling mechanisms responsible for the induced expression of interferon (IFN) genes by viral infection or double-stranded RNA (dsRNA) are not well understood. Here we investigate the role of the interferon-induced dsRNA-dependent protein kinase PKR in the regulation of IFN induction. Biological activities attributed to PKR include regulating protein synthesis, mediating IFN actions, and functioning as a possible tumor suppressor. Since binding of dsRNA is required for its activation, PKR has been considered as a candidate signal transducer for regulating IFN expression. To examine this role of PKR, loss-of-function phenotypes in stable transformants of promonocytic U-937 cells were achieved by two different strategies, overexpression of an antisense PKR transcript or a dominant negative PKR mutant gene. Both types of PKR-deficient cells were more permissive for viral replication than the control U-937 cells. As the result of PKR loss, they also showed impaired induction of IFN-alpha and IFN-beta genes in response to several inducers--specifically, encephalomyocarditis virus, lipopolysaccharide, and phorbol 12-myristate 13-acetate. Interestingly, while IFN-alpha induction by dsRNA was impaired in PKR-deficient cells, IFN-beta induction remained intact. Loss of PKR function also resulted in decreased antiviral activity as elicited by IFN-alpha and, to a greater extent, by IFN-gamma. These results implicate PKR in the regulation of several antiviral activities.

Tumour necrosis factor enhances induction by beta-interferon of a ubiquitin cross-reactive protein.

Tumour necrosis factor alpha (TNF-alpha) elicited an antiviral response in some cell lines (MG-63 and HEp-2) but not in others (MDBK). Cell lines that generated an antiviral response to TNF-alpha also showed induction of a 15K protein which shared sequence homology with ubiquitin and reacted with an antibody to ubiquitin. This ubiquitin cross-reactive protein (UCRP) had been demonstrated previously to be induced by interferon. The TNF-alpha induction of UCRP occurred at the level of transcription. TNF-alpha induction of both the antiviral state and the 15K protein was blocked by either monoclonal or polyclonal anti-beta-interferon (IFN-beta) antibody. However no measurable increase in the mRNA specific for IFN-beta was detected after TNF-alpha treatment. Nonetheless, in supernatants from cell cultures, the presence of an antiviral activity inhibitable by anti-IFN-beta antibody indicates that these cells are making IFN-beta already. We conclude that the TNF-alpha induction of antiviral activity and UCRP in cells is dependent upon the presence of constitutive low levels of IFN-beta in the responding cells. Furthermore TNF functions to enhance the existing IFN-beta activity.

Regulation of macrophage growth and antiviral activity by interferon-gamma.

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.

Application of immunoassay of encephalomyocarditis virus in cell culture with enzyme-labeled virus-specific monoclonal antibodies for rapid detection of virus, neutralizing antibodies, and interferon.

Encephalomyocarditis virus (EMCV)-specific monoclonal antibody UM 21.1 labeled with horseradish peroxidase was used to detect EMCV in L-cell monolayers. This direct enzyme immunoassay of EMCV, performed in wells of 96-well plates, could be applied for various purposes, such as early detection of virus multiplication, determination of 50% tissue culture infective doses, and rapid titration of interferon and EMCV-neutralizing antibodies. Multiplication of EMCV is indicated by a rapid increase of the absorbance values measured against EMCV-infected L cells starting as early as 4.5 h after virus inoculation. The early rise of absorbance (i.e., virus multiplication) is inhibited by interferon, allowing its rapid titration. Preincubation of the virus inoculum with neutralizing antibodies also yielded decreased absorbance values. With the latter enzyme immunoassay for neutralizing antibodies, performed after an infection period of 8 h, antibody titers measured were comparable to those obtained with a conventional plaque reduction test. We assume that similar assays could be developed for other picornaviruses (e.g., polioviruses).

Interferon inducibility and sensitivity of human teratocarcinoma-derived cell lines.

Three cell lines tera I, tera II, and PA1, derived from human teratocarcinomas were tested for their capacity to produce interferon (IFN) and for their sensitivity to both human IFN-alpha and IFN-beta. When treated with Newcastle disease virus or Sendai virus, or a synthetic polyribonucleotide, poly(rI):poly(rC), tera I cells produced no IFN and the 2',5'-oligoadenylate (2-5A) synthetase enzymatic pathway was not activated, although there was an increase in protein kinase. In contrast, tera II and PA1 cells produced IFN and both enzymatic activities were detected. IFN treatment has no effect on the growth of any of the cell lines. Tera I and PA1 cells did not develop resistance to challenge with vesicular stomatitis virus or encephalomyocarditis virus, but the growth of a type-C baboon retrovirus was inhibited. Tera II cells were protected against all three viruses. It appears that human teratocarcinoma cell lines can thus differ greatly in their ability to produce IFN and to respond to it.

Mouse peritoneal cells confer an antiviral state on mouse cell monolayers: role of interferon.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.