الفهرس 
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Abstract
Schistosomiasis caused by blood flukes of the genus Schistosoma is the second most important parasitic disease of humans. The World Health Organization has estimated that more than 207 million people are infected world wide in 74 countries and a further 700 million are at risk of this disease. While chemotherapy has succeeded in controlling the disease in many areas of the world, it has not eliminated the problem because treated individuals get reinfected and because parasites over years of intensive chemotherapy aquire resistance to reinfection. Vaccination is therefore an important approach for the longer term control and eradication of the disease. Over the past years, our laboratory at Ain Shams University has identified and cloned a large number of candidate vaccine antigens which have been, cloned, expressed and tested as recombinant protein vaccines.
The aim of the present study was to clone selected antigens in an appropriate vector for expression and testing as DNA vaccines. This vaccination strategy has a number of advantages: it is simple, and time saving in terms of not having to express and purify the recombinant protein. Equally important is the manner in which those antigens will be presented to the immune system so that they trigger the appropriate protective responses.
In this study, we sub cloned four antigen encoding sequences 14-3-3 (0.8 kb), Sm 20.8(0.8 kb),Sm cyclophilin (0.6 kb) and Sm 21.7 (0.8 kb) in the eukaryotic pSec Tag 2 vector for expression in vivo fused to an Igk- chain signal peptide that will promote secretion of the expressed antigens from the cell. The protective potential of the expressed antigens was assessed by multiple intramuscular injections as DNA vaccines in BALB/C mice. For each vaccine construct , possible effects of DNA purity on immune response and protection efficacy were assessed by injecting a subgroup of animals with ultra pure plasmid DNA prepared by purification twice over cesium chloride gradients in an ultracentrifuge. Another subgroup was injected with plasmid DNA purified by precipitation using polyethylene glycol (PEG).
The mice in each group were injected with either 100 or 300 µg pSecTag2 vaccine constructs (14-3-3 & Sm20.8) or with 100µg pSecTag2 vaccine constructs ( Sm cyclophilin & Sm21.7), and boosted by 2 additional injections over a 6 week period. A number of animals immunized with plasmid DNA purified by PEG precipitation were injected with recombinant protein rather than DNA in the last booster immunization in order to assess whether DNA vaccination will prime a humoral response to a single protein injection . The animals were then challenged with 75 cercariae 3 weeks after the last immunization. Adult worms were counted by liver perfusion 6 weeks later. Protection was calculated as percentage reduction in worm burden in comparison with a vector control group receiving pSecTag2 B vector as a negative control.
from our antigen constructs only one antigen construct, Sm14-3-3 failed to induce significant protection. We have not observed significant differences in protection between subgroups of mice receiving 100μg or 300μg DNA constructs, nor did there appear to be differences between mice subgroups injected with ultrapure gradient purified DNA and those receiving less pure PEG precipitated DNA constructs. We have therefore combined those subgroups for each antigen for statistical purposes. Significant protection levels of 38.6%, 42.2% and 63.6 % were observed in the groups of mice injected with the Sm 20.8, Sm cyclophilin and Sm21.7 constructs, respectively. Protection levels of 36.6%, 39.4% and 71.5% were observed for the subgroups of mice receiving DNA purified over CsCl gradient when injected with the Sm 20.8, Sm cyclophilin , and Sm21.7constructs, respectively. Protein boosting of animals immunized with the PEG plasmids constructs enhanced protection levels from 40.3% to 49.6% for Sm 20.8, and from 44.7% to 51.7% for Sm cyclophilin, while the protection level increased from 54% to 60.5% for Sm21.7. The booster dose of recombinant protein thus stimulated higher levels of protection in all three groups primed with DNA vaccine construct.
Humoral immune response to the 14-3-3, Sm20.8, Sm cyclophilin and Sm21.7 antigens expressed in vivo from the DNA immunization constructs was assessed using Western blots against native antigens (soluble worm antigen preparation; SWAP) and against corresponding recombinant antigens.
Compared to unimmunized mice (pre immune serum and post challenge serum of mice receiving saline injections) which did not react with the recombinant and native antigens in the Western blots, mice immunized with each of the DNA vaccine constructs generated antibody response against the corresponding recombinant protein and against the native antigen in SWAP. Antisera from Sm14-3-3 , Sm20.8, Sm cyclophilin and Sm21.7 immunized mice reacted with bands at ~27, ~ 20.8, ~17.7 and ~21.7 kDa, respectively.
In conclusion, we have cloned four candidate vaccine antigens previously identified by immunoscreening in the pSec Tag2 vector for expression in vivo with a signal tag that allows secretion from cells. Vaccination by multiple intramuscular injections with pSec Tag DNA constructs encoding tegument associated schistosome antigens is effective in triggering an immune response that provides a high level of protection in mice. The best protection level has been observed with Sm21.7 which has reached 50 -70%. Following the pSec Tag injections for Sm20.8 and Sm cyclophilin, a booster injection with the encoded recombinant protein enhanced protection from about 40% to 50%.
Our results therefore point the way for approaches for developing vaccines that rely on immune responses triggered by the expression of antigens expressed and secreted in vivo.