Expression of a functional single chain antibody on the surface of extracellular enveloped vaccinia virus as a step towards selective tumour cell targeting.

Recombinant vaccinia virus with tumour cell specificity may provide a versatile tool either for direct lysis of cancer cells or for the targeted transfer of genes encoding immunomodulatory molecules. We report the expression of a single chain antibody on the surface of extracellular enveloped vaccinia virus. The wild-type haemagglutinin, an envelope glycoprotein which is not required for viral infection and replication, was replaced by haemagglutinin fusion molecules carrying a single chain antibody directed against the tumour-associated antigen ErbB2. ErbB2 is an epidermal growth factor receptor-related tyrosine kinase overexpressed in a high percentage of human adenocarcinomas. Two fusion proteins carrying the single chain antibody at different NH2-terminal positions were expressed and exposed at the envelope of the corresponding recombinant viruses. The construct containing the antibody at the site of the immunoglobulin-like loop of the haemagglutinin was able to bind solubilized ErbB2. This is the first report of replacement of a vaccinia virus envelope protein by a specific recognition structure and represents a first step towards modifying the host cell tropism of the virus.

Dengue 2 virus enhancement in asthmatic and non asthmatic individual

During the 1981 dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) Cuban epidemic, bronchial asthma (BA) was frequently found as a personal or family antecedent in dengue hemorragic fever patients. Considering that antibody dependent enhancement (ADE) plays an important role in the etiopathogenic mechanism of DHF/DSS, we decide to study the Dengue 2 virus (D2V) capability of replication in peripheral blood leukocytes (PBL) from asthmatic patients and healthy persons. In 90% of asthmatic patients and 53.8% of control group it was obtained PBL with a significant D2V enhancing activity (X² p < 0.01). Power enhancement was higher in asthmatic group. This is the first in vitro study relating BA and the dengue 2 virus immuno enhancement. The results obtained support the role of BA as a risk factor for DHF/DSS as already described on epidemiological data.

Selective lysis of activated cells in oncorna virus infection.

The authors devised a cytotoxic assay based on cytofluorometric analysis of target surface markers in order to compare lysis exerted in vitro by cytotoxic T lymphocytes (CTLs) on different cell subsets in the context of a single lymphoid target cell population. Using this assay, the authors evaluated when oncorna virus-infected lymphocytes become a suitable target for virus-specific T cell effectors. A lymphocyte population from Moloney-murine leukaemia virus (M-MuLV)-infected (carrier) mice, in which the proliferation of selective V beta T-cell receptor (TCR) families was induced in response to Mlsa encoded antigens, was utilized as a target. The authors observed that a virus-specific T cell clone exerted lytic activity preferentially against activated cell subsets. Moreover, virus-specific CTLs generated in mixed leucocyte tumour cell cultures (MLTC) were also able to impair the concomitant anti-Mlsa response of lymphocytes from M-MuLV carrier mice. It was found that the proliferative status of oncorna virus-infected target cells played an important role in limiting the in vitro efficacy of the immune response, and it is speculated that this phenomenon might represent an in vivo escape mechanism from immunosurveillance.

Recombinant adeno-associated virus serotype 6 (rAAV2/6)-mediated gene transfer to nociceptive neurons through different routes of delivery.

BACKGROUND: Gene transfer to nociceptive neurons of the dorsal root ganglia (DRG) is a promising approach to dissect mechanisms of pain in rodents and is a potential therapeutic strategy for the treatment of persistent pain disorders such as neuropathic pain. A number of studies have demonstrated transduction of DRG neurons using herpes simplex virus, adenovirus and more recently, adeno-associated virus (AAV). Recombinant AAV are currently the gene transfer vehicles of choice for the nervous system and have several advantages over other vectors, including stable and safe gene expression. We have explored the capacity of recombinant AAV serotype 6 (rAAV2/6) to deliver genes to DRG neurons and characterized the transduction of nociceptors through five different routes of administration in mice. RESULTS: Direct injection of rAAV2/6 expressing green fluorescent protein (eGFP) into the sciatic nerve resulted in transduction of up to 30% eGFP-positive cells of L4 DRG neurons in a dose dependent manner. More than 90% of transduced cells were small and medium sized neurons (< 700 microm 2), predominantly colocalized with markers of nociceptive neurons, and had eGFP-positive central terminal fibers in the superficial lamina of the spinal cord dorsal horn. The efficiency and profile of transduction was independent of mouse genetic background. Intrathecal administration of rAAV2/6 gave the highest level of transduction (approximately 60%) and had a similar size profile and colocalization with nociceptive neurons. Intrathecal administration also transduced DRG neurons at cervical and thoracic levels and resulted in comparable levels of transduction in a mouse model for neuropathic pain. Subcutaneous and intramuscular delivery resulted in low levels of transduction in the L4 DRG. Likewise, delivery via tail vein injection resulted in relatively few eGFP-positive cells within the DRG, however, this transduction was observed at all vertebral levels and corresponded to large non-nociceptive cell types. CONCLUSION: We have found that rAAV2/6 is an efficient vector to deliver transgenes to nociceptive neurons in mice. Furthermore, the characterization of the transduction profile may facilitate gene transfer studies to dissect mechanisms behind neuropathic pain.

Interleukin-28B polymorphisms, IP-10, viral load and age predict the outcome of therapy in genotype 1 hepatitis C virus under real-life conditions

Background and Aims: IL28B polymorphisms, interferon (IFN)-gamma inducible protein-10 (IP-10) levels and the homeostasis model assessment of insulin resistance (HOMA-IR) score have been reported to predict rapid (RVR) and sustained (SVR) virological response in chronic hepatitis C (CHC), but it is not known whether these factors represent independent, clinically useful predictors. The aim of the study was to assess factors (including IL28B polymorphisms, IP-10 levels and HOMA-IR score) independently predicting response to therapy in CHC under real life conditions.Methods: Multivariate analysis of factors predicting RVR and SVR in 280 consecutive, treatment-naive CHC patients treated with pegylated IFN alpha and ribavirin in a prospective multicenter study.Results: Independent predictors of RVR were HCV RNA < 400,000 IU/ml (OR11.37; 95% CI 3.03-42.6), rs12980275 AA (vs. AG/GG) (OR 7.09; 1.97-25.56) and IP-10 (OR 0.04; 0.003-0.56) in HCV genotype 1 patients and lower baseline γ-glutamyl-transferase levels (OR = 0.02; 0.0009-0.31) in HCV genotype 3 patients. Independent predictors of SVR were rs12980275 AA (OR 9.68; 3.44-27.18), age < 40 yrs (OR = 4.79; 1.50-15.34) and HCV RNA < 400,000 IU/ml (OR 2.74; 1.03-7.27) in HCV genotype 1 patients and rs12980275 AA (OR = 6.26; 1.98-19.74) and age < 40 yrs (OR 5.37; 1.54-18.75) in the 88 HCV genotype 1 patients without a RVR. RVR was by itself predictive of SVR in HCV genotype 1 patients (32 of 33, 97%; OR 33.0; 4.06-268.32) and the only independent predictor of SVR in HCV genotype 2 (OR 9.0, 1.72-46.99; p=0.009) or 3 patients (OR 7.8, 1.43-42.67; p=0.01).Conclusions: In HCV genotype 1 patients, IL28B polymorphisms, HCV RNA load and IP-10 independently predict RVR. The combination of IL28B polymorphisms, HCV RNA level and age may yield more accurate pretreatment prediction of SVR. HOMA-IR score is not associated with viral response.

Mayaro virus proteins

Mayaro virus was grown in BHK-21 cells and purified by centrifugation in a potassium-tartrate gradient (5-50%). The electron microscopy analyses of the purified virus showed an homogeneous population of enveloped particles with 69 ñ 2.3 nm in diameter. Three structural virus proteins were identified and designated pl, p2 and p3. Their average molecular weight were p1, 54 KDa; p2, 50 KDa and p3, 34 KDa. In Mayaro virus infected. Aedes albopictus cells and in BHK-21 infected cells we detected six viral proteins, in wich three of them are the structural virus proteins and the other three were products from processing of precursors of viral proteins, whose molecular weights are 62 KDa, 64 KDa and 110 KDa. The 34 KDa protein was the first viral protein sinthesized at 5 hours post-infection in both cell lines studied.

Prevalence of antibodies to Hepatitis C virus in populations at low and high risk for sexually transmited diseases in Rio de Janeiro

In order to investigate the sexual transmission of the Hepatitis C Virus (HCV), the prevalence of specific antibodies in populations at high and low risk for sexually transmitted diseases (STDs) was evaluated. The population at low risk for STDs was composed of persons who voluntarity donated blood at the Hospital Universitário Clementino Fraga Filho (HUCFF) between July and November, 1990 (n = 2494). The population at high risk for STDs was drawn from an ongoing study on the natural history of Human Immunodeficiency Virus (HIV) infection (n = 210, 187 with sexual risk factors for HIV infection). All samples were screened using a first generation ELISA. Repeat reactive samples were then tested in a second generation RIBA. For all ELISA positive samples, two sex and age-matched ELISA negative controls were selected. Data pertaining to the presence of antibodies to the Hepatitis B core antigen (anti-HBC antibodies) and to Treponema pallidum were abstracted from the medical records. The prevalence of RIBA 2 confirmed HCV infection among the blood donors was 2.08%, which is well above the reported prevalence in similar populations from developed western countries. Among the HIV infected homosexuals, the encountered prevalence was 7.96% (p < 0.0005). For the whole group with sexually acquired HIV infection, the prevalence was 8.02% (p < 0.000005). Anti-HBc antibodies were more frequently present in anti-HCV RIBA-2 confirmed positive blood donors than in controls (p < 0.001). 33.3% of the HCV-positive blood donors and 11.04% controls were found to be anti-HBc positive (p < 0.0005). As for the FTA-ABs, 17.6% HCV-positive donors and 4,9% controls were positive (p < 0.01). 5.9% samples from blood donors were both anti-HBc and FTA-ABS positive, whereas none of the controls reacted in both tests (p < 0.05). The association between HCV, Hepatitis B infection and syphilis in individuals at low risk for parenterally transmitted diseases suggests that sexual transmission contributes to the maintenance of the endemicity of HCV in the local population.

Ultrastructural aspects of virus replication in one fatal case and several other isolates from a dengue type 2 outbreak in Rio de Janeiro

Dengue virus replication in mosquito cell cultures was observed by electron microscopy in one fatal and 40 classical isolates from a dengue type 2 outbreak in Rio de Janeiro and compared with the prototype New Guinea C strain. All the Brazilian isolates presented, beside the classical structured dengue virus particles, fuzzy coated virus-like particles, never observed in thereferencial New Guinea C virus strain. more numerous DEN-2 virus particles, fuzzy coated virus-like particles, defective virus particles and smooth membrane structures inside the rough endoplasmic reticulum characterized the unique fatal isolate examined.

Targeting mitochondria in the infection strategy of the hepatitis C virus.

Hepatitis C virus (HCV) infection induces a state of oxidative stress more pronounced than that observed in many other inflammatory diseases. Here, we propose a temporal sequence of events in the HCV-infected cell whereby the primary alteration consists of a release of Ca(2+) from the endoplasmic reticulum, followed by uptake into mitochondria. This ensues successive mitochondrial dysfunction leading to the generation of reactive oxygen species and a progressive metabolic adaptive response. Evidence is provided for a positive feed-back mechanism between alterations of calcium and redox homeostasis. This likely involves deregulation of the mitochondrial permeability transition and induces progressive dysfunction of cellular bioenergetics. Pathogenetic implications of the model and new opportunities for therapeutic intervention are discussed. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.

Studies on the replication of Mayaro virus grown in interferon treated cells

Mayaro virus grown in interferon treated infected cells has been characterized with regard to its ability to replicate in vertebrate (TC7) and invertebrate (Aedes albopictus) cells. Virus purified from interferon treated TC7 cells adsorbs and penetrates to the same extent as the control virus. During infection, these virus particles caused inhibition of host protein synthesis and synthesized the same spectrum of viral proteins as normal virus. This population however, was apparently more sensitive to interferon treatment. Electron microscopy of TC7 cells showed the presence of numerous aberrant virus particles budding from the plasma membrane.

Effects of mycophenolic acid on human immunodeficiency virus infection in vitro and in vivo.

Mycophenolic acid, a selective inhibitor of the de novo synthesis of guanosine nucleotides in T and B lymphocytes, has been proposed to inhibit human immunodeficiency virus (HIV) replication in vitro by depleting the substrate (guanosine nucleotides) for reverse transcriptase. Here we show that mycophenolic acid induced apoptosis and cell death in a large proportion of activated CD4+ T cells, thus indicating that it may inhibit HIV infection in vitro by both virological mechanisms and immunological mechanisms (depletion of the pool of activated CD4+ T lymphocytes). Administration of mycophenolate mophetil, the ester derivate of mycophenolic acid, to HIV-infected subjects treated with anti-retroviral therapy and with undetectable viremia resulted in the reduction of the number of dividing CD4 + and CD8+ T cells and in the inhibition of virus isolation from purified CD4+ T-cell populations. Based on these results, the potential use of mycophenolate mophetil in the treatment of HIV infection deserves further investigation in controlled clinical trials.