Yolanta Fishman

Cloning of the koi herpesvirus (KHV) gene encoding thymidine kinase and its use for a highly sensitive PCR based diagnosis.

BackgroundOutbreaks with mass mortality among common carp Cyprinus carpio carpio and koi Cyprinus carpio koi have occurred worldwide since 1998. The herpes-like virus isolated from diseased fish is different from Herpesvirus cyprini and channel catfish virus and was accordingly designated koi herpesvirus (KHV). Diagnosis of KHV infection based on viral isolation and current PCR assays has a limited sensitivity and therefore new tools for the diagnosis of KHV infections are necessary.ResultsA robust and sensitive PCR assay based on a defined gene sequence of KHV was developed to improve the diagnosis of KHV infection. From a KHV genomic library, a hypothetical thymidine kinase gene (TK) was identified, subcloned and expressed as a recombinant protein. Preliminary characterization of the recombinant TK showed that it has a kinase activity using dTTP but not dCTP as a substrate. A PCR assay based on primers selected from the defined DNA sequence of the TK gene was developed and resulted in a 409 bp amplified fragment. The TK based PCR assay did not amplify the DNAs of other fish herpesviruses such as Herpesvirus cyprini (CHV) and the channel catfish virus (CCV). The TK based PCR assay was specific for the detection of KHV and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions. The TK based PCR was compared to previously described PCR assays and to viral culture in diseased fish and was shown to be the most sensitive method of diagnosis of KHV infection.ConclusionThe TK based PCR assay developed in this work was shown to be specific for the detection of KHV. The TK based PCR assay was more sensitive for the detection of KHV than previously described PCR assays; it was as sensitive as virus isolation which is the golden standard method for KHV diagnosis and was able to detect as little as 10 fentograms of KHV DNA corresponding to 30 virions.

The Mycobacterium tuberculosis Recombinant 27-Kilodalton Lipoprotein Induces a Strong Th1-Type Immune Response Deleterious to Protection

ABSTRACT Th1 immune response is essential in the protection against mycobacterial intracellular pathogens. Lipoproteins trigger both humoral and cellular immune responses and may be candidate protective antigens. We studied in BALB/c mice the immunogenicity and the protection offered by the recombinant 27-kDa Mycobacterium tuberculosis lipoprotein and the corresponding DNA vaccine. Immunization with the 27-kDa antigen resulted in high titers of immunoglobulin G1 (IgG1) and IgG2a with a typical Th1 profile and a strong delayed hypersensitivity response. A strong proliferation response was observed in splenocytes, and significant nitric oxide production and gamma interferon secretion but not interleukin 10 secretion were measured. Based on these criteria, the 27-kDa antigen induced a typical Th1-type immune response thought to be necessary for protection. Surprisingly, in 27-kDa-vaccinated mice (protein or DNA vaccines) challenged by M. tuberculosis H37Rv or BCG strains, there was a significant increase in the numbers of CFU in the spleen compared to that for control groups. Furthermore, the protection provided by BCG or other mycobacterial antigens was completely abolished once the 27-kDa antigen was added to the vaccine preparations. This study indicates that the 27-kDa antigen has an adverse effect on the protection afforded by recognized vaccines. We are currently studying how the 27-kDa antigen modulates the mouse immune response.

The immunogenicity of Mycobacterium paratuberculosis 85B antigen

Abstract.Mycobacterium paratuberculosis (MPT) is the etiological agent of paratuberculosis. The disease is prevalent throughout the world, and exacts a heavy financial toll. At present, the only means of controlling this disease are culling or vaccination. The existing vaccines are not very efficient and produce a long-lasting local reaction at the point of injection and induce antibodies/delayed-type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are necessary to improve the control of this disease. We have isolated, overexpressed and purified the 85B antigen of MPT, and characterized the immune response induced by this antigen in mice. Our results showed that the recombinant MPT 85B (rMPT 85B) antigen induced a high production of interferon (IFN)γ, interleukin (IL)-6, IL-10 and nitric oxide (NO). Spleen cells from mice immunized with rMPT 85B in Ribi adjuvant produced a higher level of IL-10 and NO than spleen cells of mice immunized with rMPT 85B only. In contrast, the addition of Ribi to the immunization protocol resulted in a lower amount of IFNγ released by spleen cells. The levels of spleen cells proliferation in mice vaccinated with the rMPT 85B protein alone or with rMPT 85B with Ribi adjuvant were, respectively, four times or five times greater than in the control mice. The Ribi adjuvant induced significantly higher anti-85B antibody production of all classes tested and increased the IgG1/IgG2a ratio. DTH responses in mice footpads were observed only in mice immunized with rMPT 85B emulsified in Ribi. rMPT 85B induced both a Th1 and Th2 type of immune response with the later slightly more pronounced when the vaccination protocol comprised Ribi as an adjuvant. The rMPT 85B antigen elicited a strong immune response and can be considered as a potential candidate for a future acellular vaccine.

L-Asparaginase entrapped in liposomes: Preparation and properties

L-Asparaginase has been used with remarkable success in the treatment of certain forms of leukemia [1,2]. However, a major drawback in its continued clinical application has been the frequently observed occurrence of hypersensitive reactions to the enzyme as a foreign protein [3,4]. In principle it should be possible to prevent or at least minimize the immunogenic effect of repeated administration of the enzyme by entrapping it within non-antigenic particles. The packaging of L-asparaginase in microcapsules which are immunologically inert has been described before [5,6]. However, the objection has been raised that such microcapsules contain undegradable material which will accumulate in the body. The alternative approach of using liposomes [7] as inert vehicles [8] for L-asparaginase is outlined in this communication. We describe the preparation and some of the properties of asparaginase-carrying liposomes. Of particular interest is the observation that in such liposomes the antigenic determinants of L-asparaginase are completely masked. The evaluation of the asparagine-liposomes in vivo is now in progress.

Gamma interferon and monophosphoryl lipid A-trehalose dicorynomycolate are efficient adjuvants for Mycobacterium tuberculosis multivalent acellular vaccine.

ABSTRACT In this study, we examined the immunogenicity and protective efficacy of six immunodominant Mycobacterium tuberculosis recombinant antigens (85B, 38kDa, ESAT-6, CFP21, Mtb8.4, and 16kDa) in a multivalent vaccine preparation (6Ag). Gamma interferon (IFN-γ) and monophosphoryl lipid A-trehalose dicorynomycolate (Ribi) adjuvant systems were used separately or in combination for immunization with the recombinant antigens. Our results demonstrate that immunization of mice with Ribi emulsified antigens in the presence of IFN-γ (Ribi+6Ag+IFN-γ) resulted after challenge with a virulent M. tuberculosis strain in a significant reduction in the CFU counts that was comparable to that achieved with the BCG vaccine (∼0.9-log protection). Antigen-specific immunoglobulin G (IgG) titers in the Ribi+6Ag+IFN-γ-immunized mice were lower than in mice immunized with Ribi+6Ag and were oriented toward a Th1-type response, as confirmed by elevated IgG2a levels. In addition, splenocyte proliferation, IFN-γ secretion, and NO production were significantly higher in splenocytes derived from Ribi+6Ag+IFN-γ-immunized mice, whereas IL-10 secretion was decreased. These findings confirm the induction of a strong cellular immunity in the vaccinated mice that correlates well with their enhanced resistance to M. tuberculosis. The adjuvant effect of IFN-γ was dose dependent. A dose of 5 μg of IFN-γ per mouse per immunization gave optimal protection, whereas lower or higher amounts (0.5 or 50 μg/ mouse) of IFN-γ failed to enhance protection.

Immunogenicity of a 16.7 kDa Mycobacterium paratuberculosis antigen

Mycobacterium paratuberculosis (MPT), the agent of paratuberculosis is a slow growing mycobacteria that causes important economic losses mainly due to lower weight gains and drastic decrease in milk production. Existing paratuberculosis vaccines are not completely protective and induce antibodies/delayed type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are needed to improve the control of this disease. We have identified, expressed and purified a hypothetical thiol peroxidase of MPT (MPT-TP) in mice. We also characterized the immunogenicity of this antigen in mice. The recombinant MPT-TP (rMPT-TP) antigen induced a high production of IFNgamma, IL-6, and NO and a low production of IL-10 by spleen cells of immunized mice. Addition of Ribi adjuvant to rMPT-TP resulted in lower IFNgamma secretion and higher NO production in spleen cells. A similar level of proliferation of spleen cells exposed to rMPT-TP was found in immunized groups (rMPT-TP and rMPT-TP emulsified in Ribi). DTH responses in mice footpads were observed only in mice immunized with rMPT-TP emulsified in Ribi. Addition of Ribi adjuvant clearly induced a significantly higher anti-rMPT-TP antibody production of all classes tested and decreased the IgG1/IgG2a ratio. MPT-TP demonstrated antigenic characteristics that make this antigen a potential component in the development of a future subunit vaccine against paratuberculosis.

Molecular Characterization of Borrelia persica, the Agent of Tick Borne Relapsing Fever in Israel and the Palestinian Authority

The identification of the Tick Borne Relapsing Fever (TBRF) agent in Israel and the Palestinian Authority relies on the morphology and the association of Borrelia persica with its vector Ornithodoros tholozani. Molecular based data on B. persica are very scarce as the organism is still non-cultivable. In this study, we were able to sequence three complete 16S rRNA genes, 12 partial flaB genes, 18 partial glpQ genes, 16 rrs-ileT intergenic spacers (IGS) from nine ticks and ten human blood samples originating from the West Bank and Israel. In one sample we sequenced 7231 contiguous base pairs that covered completely the region from the 5′end of the 16S rRNA gene to the 5′end of the 23S rRNA gene comprising the whole 16S rRNA (rrs), and the following genes: Ala tRNA (alaT), Ile tRNA (ileT), adenylosuccinate lyase (purB), adenylosuccinate synthetase (purA), methylpurine-DNA glycosylase (mag), hypoxanthine-guanine phosphoribosyltransferase (hpt), an hydrolase (HAD superfamily) and a 135 bp 5′ fragment of the 23S rRNA (rrlA) genes. Phylogenic sequence analysis defined all the Borrelia isolates from O. tholozani and from human TBRF cases in Israel and the West Bank as B. persica that clustered between the African and the New World TBRF species. Gene organization of the intergenic spacer between the 16S rRNA and the 23S rRNA was similar to that of other TBRF Borrelia species and different from the Lyme disease Borrelia species. Variants of B. persica were found among the different genes of the different isolates even in the same sampling area.

Mitogenicity of the Recombinant Mycobacterial 27-Kilodalton Lipoprotein Is Not Connected to Its Antiprotective Effect

ABSTRACT We reported previously that even though immunization with the recombinant mycobacterial 27-kDa lipoprotein (r27) induced a Th1-type response in mice, the vaccinated mice became more susceptible to challenge with Mycobacterium tuberculosis. In this study we show that r27 stimulates naive splenocytes to proliferate. Acylation of r27 was crucial for this effect, since a nonacylated mutant of r27, termed r27ΔSP, failed to stimulate splenocytes either in vitro or in vivo. Depletion experiments indicated that only B cells were proliferating in a T-cell-independent manner. We also found that r27 is recognized by TLR2, which is involved in mitogenic stimulation. Interestingly, r27 but not r27ΔSP induced high gamma interferon levels in splenocyte supernatants, whereas no significant interleukin-2 levels were detected. Since B-cell polyclonal activation might aggravate pathogen infection, we asked whether the antiprotective effect of the r27 lipoprotein is associated with its mitogenicity. We showed that, as in the case of r27, immunization of mice with the nonmitogenic r27ΔSP lipoprotein resulted in increased M. tuberculosis multiplication. We conclude that the antiprotective effect of the r27 lipoprotein must be linked to properties of the polypeptide portion of the lipoprotein rather than to its lipid moiety and its mitogenicity.

Helicobacter pylori DNA in dental plaques, gastroscopy, and dental devices.

The role of dental plaque in the transmission of Helicobacter pylori (Hp) is unclear due to variability in the detection rates and techniques used. We used nested PCR to estimate the incidence of Hp in dental plaques of 24 dental hygienists. We found an unexpectedly high incidence (50%) of Hp DNA in dental plaques using sterilized dental probes. Additional treatment of sonication and SDS wash prior to sterilization of dental probes reduced the incidence to 13%. We used the treated probes to assess Hp presence in plaque samples of 47 patients visiting the dental clinic for teeth cleaning. Hp DNA was detected in 24% of cases. Since these data may reflect instrument contamination, we tested dental probes, endoscopes, and endoscopy forceps and found that 12.5–37.5% of them were contaminated. Consequently, dental plaques may be a candidate reservoir for Hp, medical equipment may contribute to Hp transmission, and sample collection techniques can bias the true prevalence of Hp in a population.