Nadav Orr

Using high titer West Nile intravenous immunoglobulin from selected Israeli donors for treatment of West Nile virus infection

BackgroundWest Nile Virus (WNV) is endemic in Israel and a significant level of antibodies is present in the population due to natural exposure. Anecdotal cases suggested that the presence of anti-WNV antibodies in intravenous immunoglobulin (IVIG) from Israeli donors (IVIG-IL) assisted the recovery of patients with severe WNV infection.MethodsTo enhance the therapeutic efficacy of IVIG-IL against WNV infection, OMRIX Biopharmaceuticals, Israel, have developed a strategy for selection of plasma units from a 10% fraction of Israeli blood donors with anti-WNV antibodies. Positive units were processed into pharmaceutical grade WNV IVIG (WNIG). Following inoculation with WNV, mice received i.p. injections of different doses (0.01–8 mg/mouse) of IVIG-IL or WNIG, according to the specific experimental protocol.ResultsWNIG was about 10 times more potent (per gr of IgG) than was regular IVIG-IL when tested by ELISA and neutralization assays. In a mouse lethal WNV infection model, prophylactic treatment with WNIG was at least 5–10-fold more potent as compared to treatment with IVIG-IL. Treatment with WNIG during active encephalitis, three or four days following WNV infection, had a significant protective effect. WNIG was also very effective in protecting immunosuppressed mice. Indeed, treatment of dexamethasone-immunosuppressed mice with 0.2 or 1.0 mg WNIG 4 h after virus infection, led to 100% survival.ConclusionIVIG produced from selected plasma donated in WNV endemic regions can be used to produce WNV IVIG with superior activity for therapeutic and prophylactic measures.

Community-Based Safety, Immunogenicity, and Transmissibility Study of the Shigella sonnei WRSS1 Vaccine in Israeli Volunteers

ABSTRACT We describe the first community-based evaluation of Shigella sonnei strain WRSS1, a live, oral candidate vaccine attenuated by a 212-bp deletion in the virG (or icsA) plasmid virulence gene. Three single-dose regimens of WRSS1 (5 × 103 CFU, 2 × 104 CFU, and 4 × 105 CFU) were tested with cohorts of 15 adult volunteers. The vaccine was generally well tolerated at the 103- and 104-CFU doses. There were no fevers and there was one report of moderate diarrhea in 30 vaccinees; five additional vaccinees reported mild diarrhea. At the 105-CFU dose, there were two reports of low-grade fevers and four reports of moderate diarrhea. The geometric means for immunoglobulin A (IgA) antibody-secreting cells (ASC) against lipopolysaccharide (LPS) were 30, 75, and 193 ASC per 106 peripheral blood mononuclear cells (PBMC) for the 103-, 104-, and 105-CFU doses, respectively. The IgG means were 40, 46, and 135 ASC per 106 PBMC, respectively. The 104-CFU dose of WRSS1 gave the best balance of safety and immunogenicity, since all vaccinees had a significant IgA ASC response and 73% had a response of more than 50 ASC. The anti-LPS seroconversion rate (threefold) for IgA was 60% and the IgG rate was 27% for the 104-CFU cohort. Each vaccinee and a cohabitating household contact delivered daily perianal stool swabs for bacteriological culture. WRSS1 colonized vaccinees for a median of 5 days, and one individual excreted WRSS1 intermittently for 23 days. None of the 45 household contacts were colonized with WRSS1 after a cumulative 192 days of cohabitation with colonized vaccinees, suggesting that adventitious vaccine spread was not common in the community setting.

Clinical and Immune Responses after Revaccination of Israeli Adults with the Lister Strain of Vaccinia Virus

BACKGROUND During the winter of 2002-2003, the Israeli health authorities launched a campaign to vaccinate first responders against smallpox. METHODS In an open study, 159 healthy, preimmunized adults, 24-52 years old, who participated in the campaign were vaccinated with the Lister strain of vaccinia virus by the multipuncture technique. The safety, immunogenicity, and reactogenicity of the vaccine were assessed. RESULTS Successful vaccination rates were 61% and 56%, on the basis of clinical take and seroconversion, respectively. Adverse events among the vaccinees were minor. Seventy-nine (88%) of the 90 vaccinees with clinical take also seroconverted ( kappa =0.779). The level of preexisting antibodies inversely correlated with the rates of clinical take and seroconversion (P</=.0098). In the group of vaccinees with the lowest preexisting levels of antibodies, 89% and 86% developed clinical take or seroconverted, respectively. The time since last vaccination was significantly associated with the rates of clinical take and seroconversion (P</=0.001). CONCLUSIONS These rates of successful vaccination in previously immunized individuals are consistent with the historical experience of use of this vaccine in Israel. The rate of occurrence and the severity of local and other reactions in the vaccinees were within the expected range. Levels of preexisting antibodies and the time since last vaccination played a major role in determining success rates.

Safety and Immunogenicity of Two Different Lots of the Oral, Killed Enterotoxigenic Escherichia coli-Cholera Toxin B Subunit Vaccine in Israeli Young Adults

ABSTRACT Enterotoxigenic Escherichia coli (ETEC) is one of the leading causes of diarrhea among Israeli soldiers serving in field units. Two double-blind placebo-controlled, randomized trials were performed among 155 healthy volunteers to evaluate the safety and immunogenicity of different lots of the oral, killed ETEC vaccine consisting of two doses of whole cells plus recombinantly produced cholera toxin B subunit (rCTB). The two doses of vaccine lot E005 and the first dose of vaccine lot E003 were well tolerated by the volunteers. However, 5 (17%) vaccinees reported an episode of vomiting a few hours after the second dose of lot E003; none of the placebo recipients reported similar symptoms. Both lots of vaccine stimulated a rate of significant antibody-secreting cell (ASC) response to CTB and to colonization factor antigen I (CFA/I) after one or two doses, ranging from 85 to 100% and from 81 to 100%, respectively. The rate of ASC response to CS2, CS4, and CS5 was slightly lower than the rate of ASC response induced to CTB, CFA/I, and CS1. The second vaccine dose enhanced the response to CTB but did not increase the frequencies or magnitude of ASC responses to the other antigens. The two lots of the ETEC vaccine induced similar rates of serum antibody responses to CTB and CFA/I which were less frequent than the ASC responses to the same antigens. Based on these safety and immunogenicity data, an efficacy study of the ETEC vaccine is under way in the Israel Defense Force.

Effective post-exposure protection against lethal orthopoxviruses infection by vaccinia immune globulin involves induction of adaptive immune response

The therapeutic potential of human vaccinia immunoglobulin (VIG) in orthopoxvirus infection was examined using two mouse models for human poxvirus, based on Ectromelia virus and Vaccinia Western Reserve (WR) respiratory infections. Despite the relatively fast clearance of human VIG from mice circulation, a single VIG injection protected immune-competent mice against both infections. Full protection against lethal Ectromelia virus infection was achieved by VIG injection up to one day post-exposure, and even injection of VIG two or three days post-infection conferred solid protection (60-80%). Nevertheless, VIG failed to protect VACV-WR challenged immune-deficient mice, even though repeated injections prolonged SCID mice survival. These results suggest the involvement of host immunity in protection. VIG provides the initial protective time-window allowing induction of the adaptive response required to achieve complete protection. Additionally, VIG can be administered in conjunction with active Vaccinia-Lister vaccination. Vaccine efficiency is not impaired, providing a non-prohibitive VIG dose is used. Thus, VIG can be used as a prophylactic measure against post-vaccinal complications but could also serve for post-exposure treatment against smallpox.

Identification of Two Distinct Populations of Protein Kinase C in Rat Brain Membranes

Abstract: The regulatory enzyme protein kinase C (PKC) is proposed to be activated on its translocation from the cytosol to the membrane. However, a portion of the native activity is always associated with the membrane fraction. Using a noninvasive procedure to extract this endogenous activity from rat brain membranes, it has been possible to characterize the activity in a partially purified reconstituted system bearing resemblance to the in vivo system. Two subpopulations of membrane‐associated PKC were identified and characterized at the level of activation, inhibition, and isozyme immunologic characteristics and chromatographic properties. One peak had properties similar to those of cytosolic PKC, whereas the second population, extracted as protein–lipid complexes, had considerable constitutive activity that could be stimulated further on addition of PKC activators. This latter activity was relatively resistant to staurosporine inhibition and phorbol ester treatment, but it phosphorylated the exogenous PKC substrates, histone 1 and the epidermal growth factor receptor peptide KTRLRR. The constitutive activity was totally dependent on its endogenous associated lipids coextracted by the solubilization procedure. The ratio between these two populations was ontogenetically regulated and modulated by phorbol ester treatment, suggesting that different PKC populations may serve unique functions in the rat brain regulated by the lipid environment. Analyses of the phospholipids extracted in these protein–lipid complexes showed differences in the major classes correlating to age. However, apart from a markedly lower cholesterol content in these complexes, no direct relationship between a specific lipid composition and the amount of constitutive PKC activity was evident.

Application of high-pressure to subfractionate membrane protein-lipid complexes: A case study of protein kinase C

Current procedures for solubilization of membrane proteins involve the use of detergents. A procedure using high hydrostatic pressures without detergent has been applied in this study to subfractionate membrane proteins and their endogenously associated lipids. Rat brain membrane preparations were suspended in hypotonic buffer containing the membrane fluidizer benzyl alcohol in a sealed pressure cell and subjected to hydrostatic pressures of up to 1500 atmospheres (approx 22,000 psi) in a French press. Under these conditions, specific membrane proteins including protein kinase C, phospholipase A2, calmodulin-binding proteins, G-proteins, and microtubule-associated proteins all coextracted and were associated to lipid particles, suggesting inherent physical contact. Two populations of membrane-associated protein kinase C were identified according to molecular weight estimations. The first coeluted with the lipid particles composed predominantly of phospholipids, while the second contained much less lipid and was similar to the soluble monomer, i.e., cytosolic protein kinase C. This procedure provides an important technique for selective subfractionation of membrane proteins in their native lipid environment which could be used for structure-function studies.

Characterization and Quantitative Analysis of Serum IgG Class and Subclass Response to Shigella sonnei and Shigella flexneri 2a Lipopolysaccharide following Natural Shigella Infection

The IgG subclass response to Shigella sonnei and Shigella flexneri 2a lipopolysaccharide (LPS) was examined in subjects naturally exposed to these organisms. Affinity-purified LPS antibodies obtained using a column of Shigella LPS bound to epoxy-activated Sepharose 6B were used as standards to calibrate the serum antibody response to natural Shigella infection. The geometric mean concentrations of specific IgG in sera from those not exposed to Shigella organisms were 7.9 microg/mL against S. sonnei LPS and 18.6 microg/mL against S. flexneri 2a LPS. After natural exposure to S. sonnei or S. flexneri 2a, the concentrations rose to 30.3 and 127.9 microg/mL, respectively. IgG2 was the major component in the anti-S. flexneri subclass response, while the anti-S. sonnei response was dominated by IgG1. High levels of IgG1 antibodies before exposure to organisms from either Shigella serogroup correlated with a lower risk of developing symptomatic infection.

Structural distinction between soluble and particulate protein kinase C species

A number of peripheral membrane proteins functioning as regulatory enzymes are distributed between soluble and particulate fractions upon homogenization and subcellular fractionation. One such enzyme, the Ca2+/phospholipid-dependent protein kinase, protein kinase C, was analyzed in order to examine this characteristic of differential localization. The soluble and particulate forms of this enzyme were purified to relative homogeneity, and their biochemical and biophysical properties were analyzed and compared. Based on biochemical activities, the particulate form required lower phospholipid concentrations for maximal activation than for the soluble species. The particulate species had a more hydrophobic structure as demonstrated by a hydrophobic fluorescence probe, and had almost 50% more α-helical structures according to secondary structure estimation, determined from far ultra-violet-circular dichroism spectra (200–250 nm). Using Fourier transform infrared spectroscopy, specific lipid spectra were detected associated with the soluble protein kinase C species. Further analyses with a fluorescent neutral membrane probe suggested that there was more lipid associated with the purified particulate form, which was of a less mobile nature than those associated with the soluble species. These structural differences provide an explanation for the preferential localization of the enzyme and may prove to be the basis for distribution of other membrane-active peripheral membrane regulatory enzymes.

High titer human immunoglobulin as a specific therapy against West Nile virus encephalitis.

WN virus is a mosquito-borne disease found most commonly in Africa, west Asia, and the Middle East, where up to 40% of the human population possesses antibodies. It is an emerging disease in the United States, since 1999 and has spread all over the US and Canada. The virus is maintained in nature in a mosquito-bird-mosquito cycle (primarily Culex), with human horses and other animals serving as incidental hosts. WN infection in humans is usually asymptomatic or involves flu like illness but can develop to severe meningo-encephlitis, with symptoms including cognitive dysfunctions, muscle weakness, paralysis and even death. Elderly and depressed immunity factors are at greatest risk of developing severe neurological disease. Studies in animal models have enhanced significantly the understanding of the viral and host factors that determine the pathogenesis and outcome of WNV disease. Currently, vaccines are available for animal use but there is no effective anti-viral therapy or human vaccine for WNV infection. Passive administration of antibodies produced from selected donors has been showed promising results in animal models.