MABF938 Sigma-AldrichAnti-MxA, clone M143 (CL143)

Although highly pathogenic avian influenza H5N1 viruses have yet to acquire the ability to transmit efficiently among humans, the increasing genetic diversity among these viruses and continued outbreaks in avian species underscore the need for more effective measures for the control and prevention of human H5N1 virus infection. Additional small animal models with which therapeutic approaches against virulent influenza viruses can be evaluated are needed. In this study, we used the guinea pig model to evaluate the relative virulence of selected avian and human influenza A viruses. We demonstrate that guinea pigs can be infected with avian and human influenza viruses, resulting in high titers of virus shedding in nasal washes for up to 5 days postinoculation (p.i.) and in lung tissue of inoculated animals. However, other physiologic indicators typically associated with virulent influenza virus strains were absent in this species. We evaluated the ability of intranasal treatment with human alpha interferon (alpha-IFN) to reduce lung and nasal wash titers in guinea pigs challenged with the reconstructed 1918 pandemic H1N1 virus or a contemporary H5N1 virus. IFN treatment initiated 1 day prior to challenge significantly reduced or prevented infection of guinea pigs by both viruses, as measured by virus titer determination and seroconversion. The expression of the antiviral Mx protein in lung tissue correlated with the reduction of virus titers. We propose that the guinea pig may serve as a useful small animal model for testing the efficacy of antiviral compounds and that alpha-IFN treatment may be a useful antiviral strategy against highly virulent strains with pandemic potential.

Among rodents that carry hantaviruses, more males are infected than females. Male rats also have elevated copies of Seoul virus RNA and reduced transcription of immune-related genes in the lungs than females. To further characterize sex differences in antiviral defenses and whether these differences are mediated by gonadal hormones, we examined viral RNA in the lungs, virus shedding in saliva, and antiviral defenses among male and female rats that were intact, gonadectomized neonatally, or gonadectomized in adulthood. Following inoculation with Seoul virus, high amounts viral RNA persisted longer in lungs from intact males than intact females. Removal of the gonads in males reduced the amount of viral RNA to levels comparable with intact females at 40 days post-inoculation (p.i.). Intact males shed more virus in saliva than intact females 15 days p.i.; removal of the gonads during either the neonatal period or in adulthood increased virus shedding in females and decreased virus shedding in males. Induction of pattern recognition receptors (PRRs; Tlr7 and Rig-I), expression of antiviral genes (Myd88, Visa, Jun, Irf7, Ifnbeta, Ifnar1, Jak2, Stat3, and Mx2), and production of Mx protein was elevated in the lungs of intact females compared with intact males. Gonadectomy had more robust effects on the induction of PRRs than on downstream IFNbeta or Mx2 expression. Putative androgen and estrogen response elements are present in the promoters of several of these antiviral genes, suggesting the propensity for sex steroids to directly affect dimorphic antiviral responses against Seoul virus infection.

The fact that adenoviral vectors activate innate immunity and induce type I IFNs has not been fully appreciated in the context of cancer gene therapy. Type I IFNs influence different aspects of human immune response and are believed to be crucial for efficient tumor rejection. We performed transcriptional profiling to characterize the response of cutaneous lymphomas to intralesional adenovirus-mediated IFN-gamma (Ad-IFN-gamma) gene transfer. Gene expression profiles of skin lesions obtained from 19 cutaneous lymphoma patients before and after treatment with Ad-IFN-gamma revealed a distinct gene signature consisting of IFN-gamma- and numerous IFN-alpha-inducible genes (type II- and type I-inducible genes, respectively). The type I IFN response appears to have been induced by the vector itself, and its complexity, in terms of immune activation, was potentiated by the IFN-gamma gene insert. Intralesional IFN-gamma expression together with the induction of a combined type I/II IFN response to Ad-IFN-gamma gene transfer seem to underlie the objective (measurable) clinical response of the treated lesions. Biological effects of type I IFNs seem to enhance those set in motion by the transgene, in our case IFN-gamma. This combination may prove to be of therapeutic importance in cytokine gene transfer using Ads.

Imiquimod, an immune response modifier that is used topically to treat different types of skin cancer, induces the production of proinflammatory cytokines that stimulate an antitumor immune response. We assessed characteristics of the imiquimod-induced immune activation in epithelial and lymphoproliferative neoplasias of human skin. We focused on plasmacytoid predendritic cells (PDCs), the primary producer of interferon alpha (IFN-alpha) after imiquimod activation in vitro.We used Affymetrix oligonucleotide arrays to compare gene expression profiles from tumors from 16 patients, 10 with superficial basal cell carcinomas (sBCCs), five with cutaneous T-cell lymphomas (CTCLs), and one with Bowen's disease, before and after topical imiquimod treatment. We used quantitative immunohistochemistry with PDC-specific antibodies against BDCA-2 and CD123 to characterize the PDC population before and after imiquimod treatment in these specimens. Activation status of PDCs from four sBCC patients was assessed by intracellular IFN-alpha staining and flow cytometry.Expression of various IFN-alpha-inducible genes (e.g., CIG5, G1P2, OASL, IFIT1, STAT1, IFI35, OAS1, ISG20, MxA, and IRF7), the so-called IFN-alpha signature, was increased similarly in both sBCC and CTCL lesions after imiquimod treatment. PDCs were recruited and activated in both lesion types, and they produced IFN-alpha after imiquimod treatment in vivo (mean percentage of PDCs producing IFN-alpha = 14.5%, 95% confidence interval [CI] = 4.9% to 24%; range = 3.3%-27%, n = 4 lesions). Imiquimod induced similar immune activation patterns in all three diseases, and these patterns were associated with the number of PDCs recruited to the treatment site. Two imiquimod-treated sBCC patients who did not mount an inflammatory response to imiquimod and whose lesions lacked the IFN-alpha signature after treatment had fewer PDCs in treated lesions compared with other treated patients with such a response.Imiquimod induces immune activation patterns that relate to the number of the PDCs recruited to the treatment site, thus supporting the role of PDC in responsiveness to imiquimod in humans.

To define domains of the human MxA GTPase involved in GTP hydrolysis and antiviral activity, we used two monoclonal antibodies (mAb) directed against different regions of the molecule. mAb 2C12 recognizes an epitope in the central interactive region of MxA, whereas mAb M143 is directed against the N-terminal G domain. mAb 2C12 greatly stimulated MxA GTPase activity, suggesting that antibody-mediated crosslinking enhances GTP hydrolysis. In contrast, monovalent Fab fragments of 2C12 abolished GTPase activity, most likely by blocking intramolecular interactions required for GTPase activation. Interestingly, intact IgG molecules and Fab fragments of 2C12 both prevented association of MxA with viral nucleocapsids and neutralized MxA antiviral activity in vivo. mAb M143 had no effect on MxA function, indicating that this antibody binds outside functional regions. These data demonstrate that the central region recognized by 2C12 is critical for regulation of GTPase activity and viral target recognition.

Measles virus (MV)-specific transcription in human brain cells is characterized by particularly low abundances of the distal mRNAs encoding the MV envelope proteins. Similar transcriptional restrictions of the closely related vesicular stomatitis virus have been observed in mouse fibroblasts constitutively expressing the interferon-inducible MxA protein (P. Staeheli and J. Pavlovic, J. Virol. 65:4498-4501, 1991). We found that MV infection of human brain cells is accompanied by rapid induction and high-level expression of endogenous MxA proteins. After stable transfection of MxA, human glioblastoma cells (U-87-MxA) released 50- to 100-fold less infectious virus and expression of viral proteins was highly restricted. The overall MV-specific transcription levels were reduced by up to 90%, accompanied by low relative frequencies of the distal MV-specific mRNAs. These restrictions were linked to an inhibition of viral RNA synthesis and not to a decreased stability of the viral RNAs. Our results indicate that expression of MxA is associated with transcriptional attenuation of MV in brain cells, thus probably contributing to the establishment of persistent MV central nervous system infections. In addition, the mechanism of MxA-dependent resistance against MV infection, in contrast to that of vesicular stomatitis virus, is cell type specific, because an inhibition of MV glycoprotein synthesis independent of transcriptional alterations was observed in MxA-transfected human monocytes (J. J. Schnorr, S. Schneider-Schaulies, A. Simon-Jödicke, J. Pavlovic, M. A. Horisberger, and V. ter Meulen, J. Virol. 67: 4760-4768, 1993).

Interferon-alpha/beta induces in cells from mice carrying the influenza virus resistance allele Mx+ the synthesis of a unique 75,000-dalton protein (designated here protein Mx) that was not detectable in interferon-treated cells from Mx- mice lacking the resistance allele (Horisberger, M. A., Staeheli, P., and Haller, O. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1910-1914). We have immunized Mx- BALB/c mice with extracts of interferon-treated cells from congenic Mx+ BALB X A2G-Mx mice. Resulting hyperimmune sera immunoprecipitated a single interferon-inducible 75,000-dalton protein of Mx+ embryo cells. Sera did not react with any interferon-inducible protein of Mx- cells. Synthesis of the immunoprecipitable 75,000-dalton protein Mx was induced by interferon-alpha/beta but not by interferon-gamma in Mx+ cells of diverse genetic backgrounds. Monoclonal antibodies to protein Mx were selected in similar immunoprecipitation assays. One discrete in vitro translation product of relevant mRNA reacted with polyclonal and monoclonal antibodies and was indistinguishable from in vivo synthesized protein Mx.