Mice orally immunized with a transgenic plant expressing the glycoprotein of Crimean-Congo hemorrhagic fever virus.

While Crimean-Congo hemorrhagic fever (CCHF) has a high mortality rate in humans, the associated virus (CCHFV) does not induce clinical symptoms in animals, but animals play an important role in disease transmission to humans. Our aim in this study was to examine the immunogenicity of the CCHFV glycoprotein when expressed in the root and leaf of transgenic plants via hairy roots and stable transformation of tobacco plants, respectively. After confirmatory analyses of transgenic plant lines and quantification of the expressed glycoprotein, mice were either fed with the transgenic leaves or roots, fed the transgenic plant material and injected subcutaneously with the plant-made CCHFV glycoprotein (fed/boosted), vaccinated with an attenuated CCHF vaccine (positive control), or received no treatment (negative control). All immunized groups had a consistent rise in anti-glycoprotein IgG and IgA antibodies in their serum and feces, respectively. The mice in the fed/boosted group showed a significant rise in specific IgG antibodies after a single boost. Our results imply that oral immunization of animals with edible materials from transgenic plants is feasible, and further assessments are under way. In addition, while the study of CCHF is challenging, our protocol should be further used to study CCHFV infection in the knockout mouse model and virus neutralization assays in biosafety level 4 laboratories.

Immune responses elicited by co-immunization of Plasmodium vivax and P. falciparum MSP-1 using prime-boost immunization strategies.

Carboxy-terminus of merozoite surface protein-1 (MSP-1(19) ) is the major protein on the surface of the plasmodial merozoite that acts as one of the most important blood-stage vaccine candidates. The present investigation was designed to evaluate the immune responses when either two recombinant antigens (rPvMSP-1(19) + rPfMSP-1(19)) or two plasmid constructs (pcDNA3.1 hygro-PvMSP-1(19) + pcDNA3.1 hygro-PfMSP-1(19)) were administered in combination at a single site in mice by using different immunization strategies (protein/protein, DNA/DNA and DNA/protein) at weeks 0, 5 and 8. All mice were monitored for the level of MSP-1(19) -specific antibody for up to 40 weeks. The inclusion of both recombinant antigens in a vaccine mixture could not inhibit induction of antibodies to the other antigen when the two recombinant antigens were combined in immunization formulation. Interestingly, antisera from immunized mice with either recombinant antigen failed to cross-react with heterologous antigen. Moreover, the results of this study showed that co-immunization with both antigens at a single site generated a substantial PvMSP-1(19) - and PfMSP-1(19) -specific antibody responses and also IFN-γ cytokine production (Th1 response) in DNA/protein prime-boost immunization strategies. The increased humoral response to PvMSP-1(19) and PfMSP-1(19) lasted nearly a year after immunization. Therefore, the results of this study are encouraging for the development of multi-species malaria vaccine based on MSP-1(19) antigen.

High frequency production of rapeseed transgenic plants via combination of microprojectile bombardment and secondary embryogenesis of microspore-derived embryos.

Transgenic doubled haploid rapeseed (Brassica napus L. cvs. Global and PF(704)) plants were obtained from microspore-derived embryo (MDE) hypocotyls using the microprojectile bombardment. The binary vector pCAMBIA3301 containing the gus and bar genes under control of CaMV 35S promoter was used for bombardment experiments. Transformed plantlets were selected and continuously maintained on selective medium containing 10 mg l(-1) phosphinothricin (PPT) and transgenic plants were obtained by selecting transformed secondary embryos. The presence, copy numbers and expression of the transgenes were confirmed by PCR, Southern blot, RT-PCR and histochemical GUS analyses. In progeny test, three out of four primary transformants for bar gene produced homozygous lines. The ploidy level of transformed plants was confirmed by flow cytometery analysis before colchicine treatment. All of the regenerated plants were haploid except one that was spontaneous diploid. High frequency of transgenic doubled haploid rapeseeds (about 15.55% for bar gene and 11.11% for gus gene) were considerably produced after colchicines treatment of the haploid plantlets. This result show a remarkable increase in production of transgenic doubled haploid rapeseed plants compared to previous studies.

Production of recombinant streptokinase in E. coli and reactivity with immunized mice.

Streptokinase (SK) is a potent plasminogen activator with widespread clinical use as a thrombolytic agent. In this study, we produce high level expression of recombinant streptokinase in E. coli by expression vector pET32a. Genomic DNA of streptokinase gene (SKC) was extracted, then amplified by polymerase chain reaction (PCR) method and sub-cloned to prokaryotic expression vector pET32a. Escherichia coli BL21 (DE3) pLysS were transformed with pET32a-skc and gene expression was induced by IPTG. The expressed protein was purified by affinity chromatography by Ni-NTA resin. High concentration of the recombinant protein obtained from the single-step purification by affinity-chromatography (Ni-NTA). The yield of recombinant streptokinase was nearly 470 mg L(-1) of initial culture. Our data showed that production of recombinant streptokinase improved by pET32a in Escherichia coli.

The profile of cytokines and IgG subclasses in BALB/c mice after immunization with Brucella ribosomal gene.

This study was evaluated the ability of DNA vaccine encoding L7/L12 protein of Brucella sp. to induce cellular and humoral immune responses in BALB/c mice and the profile of cytokines and IgG sub classes were determined. Intra muscular vaccination of mice using L7/L12 gene. Three vaccinations at 3 week intervals were performed. Cytokines and IgG subclasses were analyzed 3 week after the last DNA vaccination. Splenic lymphocytes from L7/L12pCDNA3-vaccinated mice produced high levels of IFNy (3100 pg mL(-1)) and low levels of IL-5 (300 pg mL(-1)), 3 weeks post-vaccination. The L7/L12pCDNA3 immunizations elicited high IgG2a isotype response in mice immunized. This antigen also induced IgG1 titers which were slightly lower than the IgG2a titers. Immunological analysis shows the appropriate immune response in BALB/c mice model after vaccination with L7/L12 gene. The high level of IFNgamma and low level of IL-5 in combination with high IgG2a/IgG1 ratio show the activation of Th1 cell response. The lower bacterial cfu from vaccinated mice in comparison with control groups show the efficiency of L7/L12 DNA vaccination in mice model.

Intranasal immunization of mice with VP2 DNA of human rotavirus a induces cellular and humoral immunity.

DNA vaccination using a plasmid encoding Human rotavirus A (HuRV-A) inner capsid VP2 was examined in a mouse model. BALB/c mice were immunized intranasally (i.n.) with a VP2 DNA vaccine that induced cellular and humoral immune response to HuRV-A. The increased levels of cytokines IFN-gamma and IL-4 and the production of anti-VP2 IgG antibodies were detected. In addition, splenocyte proliferation detected by MTT test was enhanced in the mice treated with the vaccine. These results may encourage the development of a HuRV-A DNA vaccine derived from the inner layer of viral capsid that can be administered i.n.

Expression and characterization of VP2 protein of human rotavirus a in a mammalian lung cell line.

A system for the expression and characterization of VP2 protein of Human rotavirus A, strain G3 was established in the mammalian lung cell line (A549). Expression of the recombinant VP2 (rVP2) protein was detected 48-72 hrs after transfection by Western blotting. The rVP2 protein expressed in A549 cells formed intracellular core-like particles (CLPs) 30 nm in diameter detected by electron microscopy (EM). These results showed that the A549 cells are suitable as efficient eukaryotic host for production of rVP2 protein.

Effect of integrated bombardment and Agrobacterium transformation system on transient GUS expression in hypocotyls of rapeseed (Brassica napus L. cv. PF704) microspore-derived embryos.

A new method for transformation of rapeseed microspore-derived embryos (MDEs), based on microwounding of MDEs by particle bombardment prior to inoculation with an Agrobacterium suspension was reported. In this study, effects of two transformation systems (integrated bombardment and Agrobacterium transformation system and the singular bombardment) on transient GUS expression in hypocotyls of rapeseed (Brassica napus L. cv. PF704) MDEs were studied. Bombardment parameters were: helium pressure, 1350 psi; distance between stopping screen and target tissue, 9 cm, gold particles size of 1.0 microm and chamber vacuum pressure, 24 in Hg. In integrated system A. tumefaciens strain AGL1 carrying the binary vector pCAMBIA3301 was used. Bombarded hypocotyls of MDEs were inoculated with Agrobacteria at OD600 = 1.0 for 10 min or OD600 = 0.25 for 24 h. Integrated transformation system increased the mean number of blue spots about 2-2.5 fold compared to singular bombardment and inoculated hypocotyls with OD600 = 1.0 for 10 min produced the highest number of blue spots per bombardment (743 +/- 23.67).

A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.

The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.

Identification of Leishmania major cysteine proteinases as targets of the immune response in humans.

In this study, we report the identification of two parasite polypeptides recognized by human sera of patients infected with Leishmania major. Isolation and sequencing of the two genes encoding these polypeptides revealed that one of the genes is similar to the L. major cathepsin L-like gene family CPB, whereas the other gene codes for the L. major homologue of the cysteine proteinase a (CPA) of L. mexicana. By restriction enzyme digestion of genomic DNA, we show that the CPB gene is present in multiple copies in contrast to the cysteine proteinase CPA gene which could be unique. Specific antibodies directed against the mature regions of both types expressed in Escherichia coli were used to analyze the expression of these polypeptides in different stages of the parasite's life cycle. Polypeptides of 27 and 40 kDa in size, corresponding to CPA and CPB respectively, were detected at higher level in amastigotes than in stationary phase promastigotes. Purified recombinant CPs were also used to examine the presence of specific antibodies in sera from either recovered or active cases of cutaneous leishmaniasis patients. Unlike sera from healthy uninfected controls, all the sera reacted with recombinant CPA and CPB. This finding indicates that individuals having recovered from cutaneous leishmaniasis or with clinically apparent disease have humoral responses to cysteine proteinases demonstrating the importance of these proteinases as targets of the immune response and also their potential use for serodiagnosis.