| Coxsackievirus B(CVB), a member of the picornavirus group, is an important human pathogen. Among the six serotypes of CVB, CVB3 is increasingly recognized as to be closely associated with acute/ chronic myocardial disease and dilated cardiomyopathy cases. Because so far there are no virus-specific preventive or therapeutic proceatures available to protect humans against CVB-induced heart diseases, research on its pathogenic mechanisms and immunity to this enterovirus has received more and more attention.CVB3 typically appears to be an icosahedral shell organized by 60 subunits containing 4 kinds of virion proteins, VP1, VP2, VP3 and VP4. As a kind of entero-infected virus, CVB3 multiplies throughout the alimentary tract and causes a variety of infections. Because of its acid stability between pH3~pH10, CVB3 could survive transit through the stomach and become implanted in the lower intestinal tract. CVB3 infects cardiac and entero cells through binding to CAR/CD55/DAF, specific receptor on the cell membranes, this binding triggers a conformational change in the virion that causes the exposure of VP1 and VP4 to the cell membranes, and then the endocytosis of the virion by cell membranes. Considering that CVB3 is an entero-infected virus and its cytopathy is closely associated with its damage to cardiac cells directly and indirectly, Induction of CVB3-specific mucosal immunity and systemic immunity appears to be an improtant strategy to prevent or perpaps treat CVB3-induced myocarditis.DNA vaccine is known for its ability to induce both humoral and cellular immune responses when delivered into animals. Its potential to generate specific CTLs affords it the opportunity to establish new preventive procedures against viral infection. To induce CVB3-specific immune responses, our previous studies have found that three immunization of 50?g DNA vaccine harboring CVB3 VP1 encoding sequence resulted in protection against CVB3 challenge. However, this DNA vaccinegenerated only IgM and IgG, no mucosal IgA could be detected. CVB3 is known to fulfill its infection via intestinal tract. Mucosal immunity specific against CVB3 is thought to play key roles in controlling early infection and elimination of viruses. Therefore induction of CVB3-specific mucosal immunity appears to be an important strategy in our study. Chitosan, the deacetylated form of chitin, is a biodegradable polysaccharide from crustscean shells. Due to its good biocompatibility and toxicity profile, it has been widely used in pharmaceutical research and in industry as a carrier for drug delivery and as biomedical material for artificial skin and wound healing bandage applications. It is also able to open the tight junctions and allow paracellular transport across the epithelium. In our study, in order to effectively induce CVB3-specific mucosal immunity, we'd like to utilize chitosan as a quickly acting absorption enhancing agent, to construct a novel chitosan-embedded gene vaccine harboring VP1 encoding sequences of CVB3 for mucosal immunization. Further more, a novel virus- like chitosan-DNA-HApLys16 vaccine was created, aimed to elevate the effecacy of chitosan-DNA vaccine by enhancing DNA expression in vivo. In this paper, in vitro and in vitro expression of chitosan-DNA, specific immunity and anti-viral immunoprotection induced by chitosan-DNA, and its potential to prevent onset of CVB3-induced myocarditis was thoroughly studied, generating results as follows: 1. Construction of a novel chitosan-DNA vaccine expressing VP1, the major structural protein of CVB3 and characterization of its propertiesThe 852bp gene coding for VP1 was obtained by RT-PCR from CVB3 propagated in Hela cells, then was digested with Hind III and Bam HI and cloned into pcDNA3 to construct pcDNA3-VP1 plasmid. Construction was confirmed by PCR, enzyme digestion and DNA sequencing. In vitro expression of VP1 was verified after transfection of Hela cells with the plasmid and ELISA detection of transgene products in the supernatant. Chitosan-DNA complex...
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