The antiviral clinical candidate 6-O-butanoyl castanospermine (MDL 28,574), an alpha-glucosidase 1 inhibitor, was examined for its effect on elementary parameters of immune function. It did not affect the mitogenic response of uninfected human mononuclear leukocytes or the detection of a range of cell surface markers, with the exception of the integrin LFA-1 (CD18/CD11a), which was reduced, after cell growth in vitro. The detection of LFA-1 was also reduced on both human and murine cells after oral administration of the compound to xenochimaeric or normal mice, respectively. Altered LFA-1 expression or function may contribute to reduced cell adhesion and the observed reduction in the in vitro allogeneic response by uninfected cells, as well as the previously described prevention of cell conjugate and HIV-induced syncytium formation.
MDL 74,695, a novel dipeptide-like compound containing the ‘difluorostatone type’ transition state mimic and a potent inhibitor of the human immunodeficiency virus (HIV) proteinase, was investigated for anti-HIV activity in vitro. The compound showed selective inhibition of both HIV-1 and HIV-2 in MT-4 cells. A potent antiviral effect against a range of clinical isolates of HIV-1 cultured in human peripheral blood mononuclear cells and primary monocytes was also demonstrated. The antiviral activity of MDL 74,695 against viruses resistant to a range of reverse transcriptase inhibitors was equivalent to the wild-type. In rats MDL 74,695 (30 mg kg −1 ) was 4.9% orally bioavailable and maintained levels above the in vitro 50% inhibitory concentration (IC 50 ) for approximately 3 h. Viruses with reduced sensitivity to MDL 74,695 and saquinavir were selected in cell culture by continuous passage in increasing drug concentrations, and first appeared after 20 and 17 passages, respectively. Amino acid changes were identified at positions 48 (glycine to valine), 50 (isoleucine to valine) and 82 (valine to either isoleucine or alanine) in various combinations for MDL 74,695-resistant viruses. For saquinavir-resistant viruses changes were identified at positions 48 (glycine to valine) and 90 (leucine to methionine). Studies using MDL 74,695, saquinavir and a third proteinase inhibitor indinavir, indicated that virus selected in the presence of MDL 74,695, with amino acid exchanges at positions 48 and 82 showed cross-resistance to saquinavir. However, viruses selected in the presence of MDL 74,695 with amino acid exchanges at positions 50 and 82 showed no significant change in sensitivity to saquinavir. Likewise, viruses selected in the presence of saquinavir with amino acid exchanges at positions 48 and 90 remained sensitive to MDL 74,695. All viruses selected after growth in the presence of either MDL 74,695 or saquinavir showed little or no resistance to indinavir.
The polymerase chain reaction (PCR) and co-cultivation were used to identify the lymphoreticular system as the site of latency of equid herpesvirus I (EHV-1). Primers for PCR were designed from aligned nucleotide sequences of the glycoprotein gB genes to amplify the same region of both the EHV-1 and EHV-4 genomes. Subsequent restriction digests using specific enzymes distinguished the amplified fragments of the EHV-1 genome from those of the EHV-4 genome. Ten weeks following an experimental infection of five ponies with EHV-1, latent virus was detected by PCR and recovered by co-cultivation, predominantly from lymphoid tissues draining the respiratory tract. Significantly, latent EHV-1 also persisted in peripheral blood leukocytes (PBL). Latent EHV-4, presumably from a preceding natural infection, was also detected in some tissues, including PBL, from all animals. Of additional interest was the recovery of EHV-1 and -4 only in the presence of the ubiquitous EHV-2.
Oral treatment of mice, cutaneously infected with herpes simplex virus type 1 (HSV-1) (strain SC16), with the α-gluco-sidase 1 inhibitor 6-O-butanoyl castanospermine (MDL 28,574) produced a significant delay in lesion development and reduced the amount of virus recovered from the brain. Virus load in the brains of mice, whose treatment started 2 days prior to infection, was reduced ˜100-fold when compared to untreated controls. Treatment initiated at the time of infection, while less effective than pre-treatment, nevertheless reduced virus recovery from the brain by 10-fold. Consistent with its antiviral activity, orally administered MDL 28,574 was rapidly incorporated by brain tissue and mice fed with compound over extended periods maintained relatively high levels of drug at this site.
When 23 ponies were infected with equid herpesvirus-1 or -4 (EHV-1 or EHV-4), nasal shedding of interferon (IFN) correlated closely with the duration of viral excretion. Equine interferon (EqIFN) was detected in the serum only from animals infected with the EHV-1 virus, and here high levels correlated with clinical symptoms of locomotor disorder and indicated a poor prognosis. Low levels of IFN were detected in explanted mononuclear cells from ponies infected with either virus.
Six Welsh Mountain pony foals were experimentally infected with a subtype 2 isolate of Equid Herpesvirus 1 (EHV-1) and subsequently examined for T cell mediated cytotoxicity against both subtypes. Cytotoxicity was not observed at 3 or 7 days after primary exposure but virus-specific, and genetically restricted, cytotoxicity of EHV-1-labelled autologous skin fibroblasts could be demonstrated 7 and 21 days after the animals were given a second exposure to live virus. Killing of subtype 2 antigen-labelled targets was more efficient than subtype 1 coated cells. This finding was paralleled by the observation that virus-neutralizing and complement-fixing antibody levels were subtype specific after the primary infection but after secondary exposure were directed against both subtypes. During primary infection the lymphocyte proliferative response to EHV-1 subtype 2 was not evident at 7 days post infection (dpi) but by 18 dpi was present in all animals. The second exposure produced an earlier (3 dpi) and larger proliferative response which was specific to the infecting isolate. The non-specific proliferative response to Concanavalin A mitogen indicated that virus infection induced a state of activation in circulating lymphocytes.
Equine sera were used to immunoprecipitate radiolabelled virus-infected cell proteins; subsequent resolution with polyacrylamide gel electrophoresis identified the EHV-1 polypeptides VP 2, 10a, 11, 13, 14, 15, 16, 20, 21 and 23a. The humoral support of ADCC by these sera was examined in vitro. Cytotoxicity could be demonstrated against both subtypes irrespective of the immunising isolate. The implications of these results are discussed.
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