S Lustig

Israel Institute of Biological Research, Wādi Hunein, Central District, Israel

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Publications (62)186.75 Total impact

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    ABSTRACT: Vaccinia virus protein A33 (A33VACV) plays an important role in protection against orthopoxviruses, and hence is included in experimental multi-subunit smallpox vaccines. In this study we show that single-dose vaccination with recombinant Sindbis virus expressing A33VACV, is sufficient to protect mice against lethal challenge with vaccinia virus WR (VACV-WR) and ectromelia virus (ECTV) but not against cowpox virus (CPXV), a closely related orthopoxvirus. Moreover, a subunit vaccine based on the cowpox virus A33 ortholog (A33CPXV) failed to protect against cowpox and only partially protected mice against VACV-WR challenge. We mapped regions of sequence variation between A33VACV and A33CPXVand analyzed the role of such variations in protection. We identified a single protective region located between residues 104--120 that harbors a putative H-2Kd T cell epitope as well as a B cell epitope - a target for the neutralizing antibody MAb-1G10 that blocks spreading of extracellular virions. Both epitopes in A33CPXV are mutated and predicted to be non-functional. Whereas vaccination with A33VACV did not induce in-vivo CTL activity to the predicted epitope, inhibition of virus spread in-vitro, and protection from lethal VACV challenge pointed to the B cell epitope highlighting the critical role of residue L118 and of adjacent compensatory residues in protection. This epitope's critical role in protection, as well as its modifications within the orthopoxvirus genus should be taken in context with the failure of A33 to protect against CPXV as demonstrated here. These findings should be considered when developing new subunit vaccines and monoclonal antibody based therapeutics against orthopoxviruses, especially variola virus, the etiologic agent of smallpox.
    Virology Journal 07/2013; 10(1):229. · 2.09 Impact Factor
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    ABSTRACT: In an event of a smallpox outbreak in humans, the window for efficacious treatment by vaccination with vaccinia viruses (VACV) is believed to be limited to the first few days post-exposure (p.e.). We recently demonstrated in a mouse model for human smallpox, that active immunization 2-3 days p.e. with either VACV-Lister or modified VACV Ankara (MVA) vaccines, can rescue animals from lethal challenge of ectromelia virus (ECTV), the causative agent of mousepox. The present study was carried out in order to determine whether a single dose of the anti-viral cidofovir (CDV), administered at different times and doses p.e. either alone or in conjunction with active vaccination, can rescue ECTV infected mice. Animals were infected intranasally with ECTV, treated on different days with various single CDV doses and monitored for morbidity, mortality and humoral response. In addition, in order to determine the influence of CDV on the immune response following vaccination, both the "clinical take", IFN-gamma and IgG Ab levels in the serum were evaluated as well as the ability of the mice to withstand a lethal challenge of ECTV. Finally the efficacy of a combined treatment regime of CDV and vaccination p.e. was determined. A single p.e. CDV treatment is sufficient for protection depending on the initiation time and dose (2.5 - 100 mg/kg) of treatment. Solid protection was achieved by a low dose (5 mg/kg) CDV treatment even if given at day 6 p.e., approximately 4 days before death of the control infected untreated mice (mean time to death (MTTD) 10.2). At the same time point complete protection was achieved by single treatment with higher doses of CDV (25 or 100 mg/kg). Irrespective of treatment dose, all surviving animals developed a protective immune response even when the CDV treatment was initiated one day p.e.. After seven days post treatment with the highest dose (100 mg/kg), virus was still detected in some organs (e.g. lung and liver) yet all animals survived, suggesting that efficacious single CDV treatment requires a potent immune system. The combination of CDV and vaccination provided no additional protection over CDV alone. Yet, combining CDV and vaccination maintained vaccination efficacy. Altogether, our data substantiate the feasibility of single post-exposure antiviral treatment to face orthopoxvirus infection.
    Virology Journal 06/2012; 9:119. · 2.09 Impact Factor
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    ABSTRACT: Smallpox vaccination might be associated with adverse reactions, ranging in severity from benign to lethal. One of the most serious complications is postvaccinal encephalitis. The aim of this study was to identify early markers for vaccinia virus (VACV) induced encephalitis. For this purpose we infected mice intracranially with VACV-Lister or VACV-WR. Histopathological analysis showed that following infection with VACV-WR tissue damage in the brain spatially and temporally correlated with virus replication, infiltration of white blood cells and apoptosis. None of the above markers was observed upon infection with the vaccine strain Lister. MMP-9 is a serum factor known for its correlation to BBB integrity and encephalitis in humans. We found that in sera of VACV-Lister infected animals, MMP-9 levels did not change throughout the infection. However, during VACV-WR infection, in the first 2 days levels of MMP-9 were significantly low than the controls and subsequently rose to levels which were significantly higher than the controls. Elevated MMP-9 was associated with damage to the brain and to BBB integrity. In conclusion, efficient virus replication in the brain causes significant brain damage followed by BBB brake-down and release of MMP-9 to the serum, markers which were not observed when the attenuated strain was examined. Thus, we suggest MMP-9 as a possible non-invasive serum indicator for encephalitis caused by VACV virus. KeywordsBlood-brain-barrier-Encephalitis-Matrix metalloproteinase-9-Smallpox-Vaccinia
    06/2010: pages 147-155;
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    ABSTRACT: Vaccinia Immune Globulin (VIG) is currently used to treat severe complications of smallpox vaccines. In this study we compare the therapeutic potential of vaccinia virus rabbit hyper immune sera (RHIS) with that of human VIG. The clearance rate of RHIS from mouse circulation is only slightly slower than that of VIG (t 1/2=10 and 7.5 days respectively). Like VIG, passively administered RHIS can protect mice against lethal respiratory and dermal Ectromelia virus (ECTV) challenge. Administration of both homologous (anti ECTV) and heterologous (anti VACV-WR or VACV-Lister) anti-sera conferred efficient protection against a subsequent lethal respiratory ECTV challenge. These observations formed the basis for passive cross protection studies against ECTV, conducted in mice. RHIS conferred better protection as compared to VIG as a result of its better specific activity which is about 100 folds higher than that of VIG, allowing for significant protection even if administered 5 days post infection. This study emphasizes the advantage of a hyper immune product and validates the potential use of VIG and other antibody based therapeutics, not only as prophylactic measures against post-vaccination complications but also for post-exposure treatment of smallpox disease. KeywordsECTV-Hairless mice-Hyper immune sera-Smallpox-Vaccinia Immune Globulin (VIG)
    06/2010: pages 253-260;
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    ABSTRACT: Immunization of BALB/c mice with vaccinia virus protein A33 (A33VACV) protects mice from intranasal challenge with the WR strain of vaccinia virus or with ectromelia virus making A33 an important candidate to be included in experimental smallpox subunit vaccines. Single vaccination with a recombinant Sindbis virus expressing A33VACV protect mice against lethal VACV-WR and ectromelia virus (ECTV) but not against the closely related cowpox virus (CPXV). Furthermore, even recombinant Sindbis virus expressing the cowpox virus A33 ortholog (A33CPXV) failed to protect either against cowpox or against VACV-WR challenge. Our attempts to map the regions which may account for this differential behavior were directed against a region of difference between the two orthologs. A stretch of 7 amino acids in A33 was mapped as important for protection which contain the following changes in A33CPXV: L112F, Q117K and L118S. This region maps to a single putative prevalent 9-mer CTL epitope with L112 as an essential anchoring residue, and a major target epitope for neutralizing antibodies encompassing L118. Vaccination with A33 harboring these individual substitutions highlighted the crucial role of L118 in induction of protective immunity. KeywordsSmallpox-A33-Cowpox-Vaccinia-Alphavirus
    06/2010: pages 115-123;
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    ABSTRACT: The Variola virus is the causative agent of smallpox disease. Accurate identification of Variola and differential diagnosis between Variola and other vesicle forming (smallpox-like) pathogens are both technically challenging and of great importance. We compiled a list of vesicle forming pathogens that are prone to be misdiagnosed as smallpox. Some of the pathogens (members of the Orthopoxvirus genus) are also genetically highly similar to Variola. We established an approach for genetic identification and differential diagnosis between the closely related Orthopoxvirus genus members. This approach couples multiplex PCR with DNA microarray as molecular means for specific and sensitive pathogen identification. It consists of PCR amplification of conserved gene fragments, followed by sequence-based specific identification on a DNA microarray platform utilizing the Arrayed Primer Extension (APEX) technique. Our prototype array – the ChiPox, is based on genes that harbor variable regions flanked by highly conserved Orthopoxvirus sequences; a pattern supporting APEX-based diagnostics. This novel approach was successfully applied for the differential identification of six members of the Orthopoxvirus genus. The ChiPox is a powerful and sensitive tool for discrimination between closely related Orthopoxvirus species, and is the first step in the establishment of a comprehensive assay for genetic discrimination of vesicle forming pathogens. KeywordsAPEX-Orthopoxvirus-Vesicle-forming pathogens-DNA microarray-Smallpox
    06/2010: pages 173-182;
  • Nir Paran, Shlomo Lustig
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    ABSTRACT: The mechanism of protection afforded by vaccinia virus (VACV) - the smallpox vaccine - is a key issue for the development of modern vaccines and countermeasures. Antibodies to VACV antigens of the extracellular virion (EV) form play a central role in protection against poxvirus diseases in animal models, and contribute to the protection of immunized humans against poxviruses. B5, a viral EV protein, is conserved among different orthopoxviruses and antibodies to B5 that protect mice against VACV challenge. Antibodies to B5 are primarily responsible for neutralization of vaccinia EVs, yet the mechanism of EV neutralization by antibodies to B5 is not fully understood. The paper under evaluation demonstrates that most of the neutralization in vitro and protection in vivo in a mouse model, by monoclonal human anti-B5 IgGs, is heavily dependent on the ability of the IgGs to bind complement (C3 and C1q). Similarly, IgGs capable of complement binding control complement-dependent cytotoxicity of VACV-infected cells. Human polyclonal antibodies induced by the smallpox vaccine were similarly dependent on complement for EV neutralization and the complement-dependent destruction of infected cells. These findings not only contribute to a better understanding of the mechanism of protection by antibodies, but might also help in the development and evaluation of newly-developed therapeutic and prophylactic antibody-based products against virulent orthopoxviruses, and for the prevention or treatment of smallpox vaccine-related post-vaccinal adverse effects.
    Expert Review of Vaccines 03/2010; 9(3):255-9. · 4.22 Impact Factor
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    ABSTRACT: Ectromelia virus, a member of the Orthopox genus, is the causative agent of the highly infectious mousepox disease. Previous studies have shown that different poxviruses induce cell-cell fusion which is manifested by the formation of multinucleated-giant cells (polykaryocytes). This phenomenon has been widely studied with vaccinia virus in conditions which require artificial acidification of the medium. We show that Ectromelia virus induces cell-cell fusion under neutral pH conditions and requires the presence of a sufficient amount of viral particles on the plasma membrane of infected cells. This could be achieved by infection with a replicating virus and its propagation in infected cells (fusion "from within") or by infection with a high amount of virus particles per cell (fusion "from without"). Inhibition of virus maturation or inhibition of virus transport on microtubules towards the plasma membrane resulted in a complete inhibition of syncytia formation. We show that in contrast to vaccinia virus, Ectromelia virus induces cell-cell fusion irrespectively of its hemagglutination properties and cell-surface expression of the orthologs of the fusion inhibitory complex, A56 and K2. Additionally, cell-cell fusion was also detected in mice lungs following lethal respiratory infection. Ectromelia virus induces spontaneous cell-cell fusion in-vitro and in-vivo although expressing an A56/K2 fusion inhibitory complex. This syncytia formation property cannot be attributed to the 37 amino acid deletion in ECTV A56.
    Virology Journal 09/2009; 6:151. · 2.09 Impact Factor
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    ABSTRACT: 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.
    Vaccine 03/2009; 27(11):1691-9. · 3.49 Impact Factor
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    ABSTRACT: Decades after the cessation of smallpox vaccination, the potential of the deliberate release of pathogenic orthopoxviruses has forced a reconsideration of using these extremely efficient human vaccines. Scenarios of sudden biothreats have prompted demand for rapidly protective vaccination. However, the feasibility of short-term vaccination (i.e., vaccination shortly before exposure) with vaccinia virus (VACV) is uncertain. We tested the rapid protective capacity of vaccines based on VACV strain Lister (VACV-Lister) and on modified VACV Ankara (MVA) in different mouse models, comparing lethal infections with VACV strain Western Reserve (VACV-WR) or ectromelia virus (ECTV). In contrast to VACV-WR challenge, we found extended incubation periods after ECTV challenge, allowing successful therapeutic immunization with VACV-Lister and MVA when applied 2-3 days after exposure. Rapid protection from respiratory tract ECTV infection was significantly affected by vaccine dose and was associated with occurrence of poxvirus-specific antibodies. Vaccinations in type I interferon receptor-deficient mice were protective, whereas recombination activating gene 1-deficient mice lacking mature T and B cells failed to mount immunity after short-term vaccination, confirming an essential role of adaptive immune responses. ECTV infection in mice models the course of human smallpox. Our data provide evidence to substantiate historical data on the usefulness of postexposure vaccination with conventional VACV and the new candidate MVA to protect against fatal orthopoxvirus infections.
    The Journal of Infectious Diseases 12/2008; 199(1):39-48. · 5.85 Impact Factor
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    Vaccine. 01/2008; 26(21):2667-2667.
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    ABSTRACT: Since smallpox eradication by the WHO during the 1980s, potency of new vaccines is compared to vaccines that were used during the eradication campaign. In this work we characterize the tail scarification technique in mice as a model for scarification in humans. Similar to humans, mice develop "clinical take" which is dependent on the vaccination dose. Appearance of anti-Vaccinia IgM is followed by IgG antibodies 10 days post scarification and lasting more then 1(1/2) years. Mice with "clinical take" are 100% protected against lethal respiratory challenge (100LD(50)) of Vaccinia WR indicating that the "clinical take" can serve as a correlate of protective immunity. Reducing the vaccination dose and using Cowpox virus as a more virulent strain, enabled us to draw the limit of the vaccine potency in mice. Similar to humans, in revaccinated mice the development of "clinical take" was inversely correlated to the level of pre-existing antibodies. These results indicate that tail scarification of mice can be used as a model for evaluation of smallpox vaccines. High correlation between "clinical take" and protective immunity allows the use of visual inspection to evaluate vaccine potency.
    Vaccine 12/2007; 25(45):7743-53. · 3.49 Impact Factor
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    ABSTRACT: Recombinant proteins are being evaluated as smallpox and monkeypox vaccines because of their perceived safety compared to live vaccinia virus. Previously, we demonstrated that three or more injections of a Ribi-type adjuvant with a combination of three proteins from the outer membranes of intracellular (L1 protein) and extracellular (A33 and B5 proteins) forms of vaccinia virus protected mice against a lethal intranasal challenge with vaccinia virus. Here, we compared several adjuvants and found that QS-21 and to a lesser extent alum+CpG oligodeoxynucleotides accelerated and enhanced neutralizing antibody responses to a mixture of L1 and A33 proteins, provided the highest ratio of IgG2a to IgG1 isotype response, and protected mice against disease and death after only two immunizations 3 weeks apart. In addition, monkeys immunized with recombinant vaccinia virus proteins and QS-21 developed neutralizing antibody to monkeypox virus and had reduced virus load, skin lesions, and morbidity compared to the non-immunized group following monkeypox virus challenge.
    Vaccine 05/2007; 25(15):2787-99. · 3.49 Impact Factor
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    ABSTRACT: Previous studies have demonstrated that dehydroepiadrosterone (DHEA) bears an immunomodulating effect, and while having no antiviral activity in vitro, is capable of protecting against lethal viral infections. In the present study the effect of DHEA on protection from stress-induced infection was examined. Mice were exposed to cold stress, isolation stress or dexamethasone injections and inoculated with the attenuated non-invasive WN-25 encephalitis virus. DHEA treatment prevented the induction of encephalitis and decreased mortality rate from 67 per cent, 75 per cent and 67 per cent to 22 per cent, 32 per cent and 11 per cent, respectively. Postponement of the onset of disease, reduced virus levels in the brain on day 7, increased mean time of death, and prevention of spleen and thymus involution was also observed. The protective effect is mediated probably by counteraction of stress-induced immunosuppression that appears during viral infections. We suggest that DHEA may act as an ‘antistress’ agent, an agent that is effective in protection against viral infection by blocking stress-induced immunosuppression.
    Stress Medicine 02/2006; 8(3):181 - 185.
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    ABSTRACT: Vaccination with vaccinia virus is carried out in order to induce protection against variola virus, the causative agent of smallpox. Serum titer of vaccinia virus-neutralizing antibodies is considered to be well-correlated with in vivo protection. Plaque reduction neutralization test (PRNT) is the gold standard for detecting and quantifying vaccinia virus-neutralizing antibodies in sera of vaccinees. However, PRNT is time and labor consuming, which does not allow large-scale screening needed for a population survey. A simplified, sensitive, standardized, reproducible and rapid method, neutralization tissue-culture enzyme immunoassay (NTC-EIA) was developed for quantitation of neutralizing antibodies against vaccinia virus. The assay consists of the following steps: neutralization of the virus with serially diluted sera, infection of cells in culture and measurement of residual virus replication using an enzyme immunoassay. The assay can be used for animal (rabbit) or human sera. Titer averages obtained using NTC-EIA were highly correlated (R2=0.9994) to those obtained using PRNT. The assay is carried out in 96-well plates and takes only 2 days to complete. With the appropriate setup, it can be automated fully to allow screening of a large number of sera.
    Journal of Virological Methods 01/2006; 130(1-2):15-21. · 1.90 Impact Factor
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    ABSTRACT: Previous studies demonstrated that antibodies to live vaccinia virus infection are needed for optimal protection against orthopoxvirus infection. The present report is the first to compare the protective abilities of individual and combinations of specific polyclonal and monoclonal antibodies that target proteins of the intracellular (IMV) and extracellular (EV) forms of vaccinia virus. The antibodies were directed to one IMV membrane protein, L1, and to two outer EV membrane proteins, A33 and B5. In vitro studies showed that the antibodies to L1 neutralized IMV and that the antibodies to A33 and B5 prevented the spread of EV in liquid medium. Prophylactic administration of individual antibodies to BALB/c mice partially protected them against disease following intranasal challenge with lethal doses of vaccinia virus. Combinations of antibodies, particularly anti-L1 and -A33 or -L1 and -B5, provided enhanced protection when administered 1 day before or 2 days after challenge. Furthermore, the protection was superior to that achieved with pooled immune gamma globulin from human volunteers inoculated with live vaccinia virus. In addition, single injections of anti-L1 plus anti-A33 antibodies greatly delayed the deaths of severe combined immunodeficiency mice challenged with vaccinia virus. These studies suggest that antibodies to two or three viral membrane proteins optimally derived from the outer membranes of IMV and EV, may be beneficial for prophylaxis or therapy of orthopoxvirus infections.
    Journal of Virology 12/2005; 79(21):13454-62. · 5.08 Impact Factor
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    ABSTRACT: The two forms of infectious vaccinia virus particles, known as intracellular mature virions and extracellular enveloped virions, are liberated by cell lysis and exocytosis, respectively. The extracellular enveloped form, which is highly resistant to antibody neutralization, contains an outer membrane surrounding an intracellular mature form. We provide evidence that complement mediates antibody-dependent lysis of the outer membrane of extracellular virus, exposing the inner infectious virus to neutralization by a second antibody. These results can help explain the disparity between the in vitro neutralizing and in vivo protective effects of antibodies to extracellular envelope proteins as well as the enhanced protection afforded by specific combinations of antibodies.
    Virology 11/2004; 328(1):30-5. · 3.37 Impact Factor
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    ABSTRACT: Infectious intracellular and extracellular forms of vaccinia virus have different outer membrane proteins, presenting multiple targets to the immune system. We investigated the immunogenicity of soluble forms of L1, an outer membrane protein of the intracellular mature virus, and of A33 and B5, outer membrane proteins of the extracellular enveloped virus. The recombinant proteins, in 10-microg amounts mixed with a Ribi- or saponin-type adjuvant, were administered subcutaneously to mice. Antibody titers to each protein rose sharply after the first and second boosts, reaching levels that surpassed those induced by percutaneous immunization with live vaccinia virus. Immunoglobulin G1 (IgG1) antibody predominated after the protein immunizations, indicative of a T-helper cell type 2 response, whereas live vaccinia virus induced mainly IgG2a, indicative of a T-helper cell type 1 response. Mice immunized with any one of the recombinant proteins survived an intranasal challenge with 5 times the 50% lethal dose of the pathogenic WR strain of vaccinia virus. Measurements of weight loss indicated that the A33 immunization most effectively prevented disease. The superiority of protein combinations was demonstrated when the challenge virus dose was increased 20-fold. The best protection was obtained with a vaccine made by combining recombinant proteins of the outer membranes of intracellular and extracellular virus. Indeed, mice immunized with A33 plus B5 plus L1 or with A33 plus L1 were better protected than mice immunized with live vaccinia virus. Three immunizations with the three-protein combination were necessary and sufficient for complete protection. These studies suggest the feasibility of a multiprotein smallpox vaccine.
    Journal of Virology 11/2004; 78(19):10230-7. · 5.08 Impact Factor
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    ABSTRACT: During the winter of 2002-2003, the Israeli health authorities launched a campaign to vaccinate first responders against smallpox. 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. 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). 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.
    The Journal of Infectious Diseases 10/2004; 190(7):1295-302. · 5.85 Impact Factor

Publication Stats

2k Citations
186.75 Total Impact Points


  • 1990–2013
    • Israel Institute of Biological Research
      Wādi Hunein, Central District, Israel
  • 2005–2007
    • National Institutes of Health
      • Laboratory of Viral Diseases
      Bethesda, MD, United States
  • 2004
    • National Institute of Allergy and Infectious Diseases
      • Laboratory of Immunoregulation
      Maryland, United States
  • 1998
    • Washington University in St. Louis
      • Department of Molecular Microbiology
      Saint Louis, MO, United States
  • 1997
    • Bar Ilan University
      • Faculty of Life Sciences
      Gan, Tel Aviv, Israel