Construction Of B Subunit Of Shiga Toxin And Coxsackievirus B3 VP1 Fusion DNA Vaccine And Study On Its Immunological Effects On Mice

Object: Coxsackievirus B3 (CVB3) is a major pathogen of human virus myocarditis. So far there are no virus-specific preventive or therapeutic procedures available to protect humans against coxsackievirus-induced myocarditis. Immunization with DNA vaccine affords an opportunity to establish a new preventive procedure against CVB3 infections. The gene encoding VP1 protein which is the major capsid protein of CVB3 is the most promising candidate of CVB3 DNA vaccines. Some studies have shown that VP1 gene vaccine could elicit humoral and cell-mediated immune responses on mice and protect mice from CVB3 challenge. However, the neutralizing antibody titers were low and the protection effect aganist CVB3 challenge was not satisfactory. Therefore, enhancing the protective effects of VP1 gene vaccine is so far a problem need to be resovled. Shiga toxin is composed of an A subunit, which mediates toxicity, and a B subunit (STxB), a nontoxic homopentameric protein responsible for toxin binding and internalization into target cells by interacting with the glycolipid Gb3 receptor. This receptor is highly expressed on various dendritic cells (DC), which are the most powerful professional antigen-presenting cells (APC). Some studies have shown that via the glycolipid Gb3, STxB induces both MHC class I-and II-restricted presentation of peptides derived from STxB-coupled full-size exogenous protein in dendritic cells and elicits strong humoral and cell-mediated immune responses without the use of adjuvant. StxB is an attractive vector for vaccine development due to its ability to target dendritic cells and to induce specific CTL and humoral immune response. It is possible that STxB fused with VP1 could enhance the effects of CVB3 DNA vaccines. Based on this view, we plan to construct the eukaryotic expressing plasmid of STxB fused with CVB3 VP1 linked by a flexible polypeptide (Linker) as a DNA vaccine and observe its immunological effects on mice. Methods: The gene fragments of STxB and STxB-Linker were amplified by PCR from the DNA of Shigella dysenteriae. The gene fragments of VP1 and Linker-VP1 were amplified by RT-PCR from the RNA of CVB3. These four gene fragments were inserted into pGEM-T vector. E.coli DH5 αwas transformed and the recombinant clones were selected. After identified by endonuclease analyses and gene sequencing, the clone vectors were cut by proper endonucleases and the inserted gene fragments were linked to pcDNA3 cut by the same endonucleases to construct the eukaryotic expressing plasmids pcDNA3/STxB-Linker-VP1, pcDNA3/STxB and pcDNA3/VP1. After transformation, the recombined plasmids were identified by endonuclease analyses. The plasmids purified with PEG wereused as vaccines. BALB/c mice were divided into 5 groups at random: pcDNA3/STxB-Linker-VP1 group, pcDNA3/VP1 group, pcDNA3/STxB and pcDNA3/VP1 group, pcDNA3/STxB group and pcDNA3 group. BALB/c mice were inoculated intramuscularly (i.m.) in tibialis anterior muscle with a dose of 100μg DNA at 3-week intervals. At 21st day after every injection, sera were collected to detect the titers of neutralizing antibodies against CVB3. Three weeks after the third immunization, mice were challenged with 5LD50 CVB3 and the number of surviving animals was monitored up to 3 weeks post infection. Results: (1) The gene fragments of STxB, STxB-Linker, VP1 and Linker-VP1 were amplified by PCR or RT-PCR. The lengths of the fragments were the same as expected. (2) The clone vectors pGEM-T/STxB, pGEM-T/STxB-Linker, pGEM-T/VP1 and pGEM-T/Linker-VP1 were constructed successfully; endonuclease analyses and sequencing showed that inserted genes were correct. (3) After these gene fragments were linked to pcDNA3, the recombined plasmids were identified by endonuclease analyses and the results were identical as expected. That suggested that the eukaryotic expressing plasmids pcDNA3/STxB, pcDNA3/VP1 and pcDNA3/ STxB-Linker-VP1 were constructed successfully. (4) PEG pured plasmids were used as DNA vaccines. The mean titers of neutralizing antibody after every immunization in pcDNA3/STxB-Linker-VP1 group were 1:10.00, 1:25.20,1:31.75, respectively; that in pcDNA3/VP1 group were 1:16.87, 1:44.90, 1:80.00, respectively; and that in pcDNA3/STxB and pcDNA3/VP1 group were 1:11.23,1:22.45,1:36.65, respectively. The mean titers of neutralizing antibody in pcDNA3 group and pcDNA3/STxB group were both lower than 1:5. The mean titers of neutralizing antibody after the third immunization in pcDNA3/ STxB-Linker-VP1 group and pcDNA3/VP1 group were significantly different by using LSD-t test (p<0.05), showing that pcDNA3/ STxB-Linker-VP1 could elicit significantly lower level of neutralizing antibody response when compared with pcDNA3/VP1. (5) Up to the 21st day after 5LD₅₀ CVB3 challenge, survival rates of pcDNA3/ STxB-Linker-VP1 group, pcDNA3/VP1 group, pcDNA3/ STxB and pcDNA3/VP1 group, pcDNA3/ STxB group and pcDNA3 group were 44%, 68%, 33%, 40% and 32%, respectively. The survival rate of pcDNA3/VP1 group was higher than those of other groups, and the difference between pcDNA3/VP1 group and pcDNA3 control group was significant byχ² test (P<0.05). Similar result was acquired between pcDNA3/VP1 group and pcDNA3/ STxB and pcDNA3/VP1 group (P<0.05). The differences between other groups were not significant by x² test (P>0.05). These results showed that pcDNA3/VP1 could protect mice from CVB3 challenge, but pcDNA3/STxB-Linker-VP1 failed to enhance this protection. Conclusion: (1) Fusion DNA vaccine pcDNA3/STxB-Linker-VP1 was constructed. (2) The neutralizing antibody activity...