The Construction Of Eukaryotic Expression Plasmid PcDNA3/VP1-hIL-2 And The Observation Of Its Protective Effect On Mice

Objective: Coxsackievirus B3(CVB3) infections are common causes of acute and chronic myocarditis in human, and so far there are no virus-specific preventive or therapeutic procedures available that protect human against coxackievirus-induced heart diseases. Immunization with DNA affords an opportunity to establish new preventive procedures against lethal CVB3 infections. According to the fact that several immunogenic epitopes are located within the major capsid protein VP1, we constructed eukaryotic expression plasmid pcDNA3/VPl. One promising approach to enhance the efficacy of DNA vaccines is the use of cytokines which can function as immunologic adjuvants when administered during the development of an immune responses to a particular antigen. Among a broad panel of cytokine, IL-2 can upregulate both humoral and cellular immune responses. The objective of our research is to construct a bicistronic plasmid pcDNA3/VPl-hIL-2 coexpressing both CVB3 VP1 and hIL-2 and investigate whether the use of plasmid-based hIL-2 can augment immune responses elicited by VP1 plasmid DNA vaccine, which will lay a foundation for constructing a safe and effective vaccine against CVB3infections.Methods: The whole segment of CMV-VP1-BGH was amplified by PCR from plasmid pcDNA3/VPl and inserted into pGEM-T Easy vector. E.coli DH5a was transformed and the recombinant clones were selected. After identified by endonuclease analyses and sequencing, PCR products were digested with Bgl II and Mun I , gel purified, and inserted into pcDNA3/hIL-2 between the Bgl II and Mun I sites to generate a coexpression plasmid pcDNA3/VPl-hIL-2. BALB/C mice were inoculated intramuscularly three times in the quadriceps at 1-week intervals. Six days after every injection, sera were obtained and analyzed for the presence of CVB3-specific neutralizing antibodies. One week after the third immunization, mice were subjected to intraperitoneal challenge with 800TCID50 CVB3 and the number of surviving animals was monitored up to 3 weeks post infection.Results: (1) A bicistronic vector, pcDNA3/VPl-hIL-2 was constructed, which can express CVB3 VP1 protein and IL-2 within the same construction under discrete transcriptional control. (2) Neutralizing antibodies were produced in sera of pcDNA3/VPl-hIL-2-immunized mice after the second inoculation and the liters of which were <1:5, 1:28.28, 1:45.95, respectively. The liters of pcDNA3/VPl + pcDNA3/hIL-2 group after each inoculation were <1:5, 1:26.39, 1:42.87. While lower levels of antibodies (1:22.97) in sera of pcDNA3/VPl -immunized mice were detectable onlyafter the last inoculation. (3) The survival rates of pcDNA3/VPl-hIL-2, pcDNA3/VPl+ pcDNA3/hIL-2, pcDNA3/VPl, pcDNA3/IL-2, pcDNA3 immunized groups and saline control group were 25 %, 10 %, 15 %, 5 %, 15 %, 10 %, respecti\Tely. The survival rates of 6 groups had no significant difference.Conclusion: (1) The eukaryotic expression plasmid pcDNA3/VPl-hIL-2 has been successfully constructed; (2) The plasmid pcDNA3/VPl-hIL-2 could express in mice and induced the mice to produce neutralizing antibodies earlier and higher than those of pcDNA3/VPl-immunized mice. (3) Administration of pcDNA3/VPl-hIL-2 showed no evident protective effect to mice challenged with subsequent lethal infection of CVB3 under the immunologic procedures used in this research.