Enhancement Of Neutralizing Antibody Response Of Coxsackievirus B3 VP1 DNA Vaccine By A Signal Peptide DNA Of Human IL-2

Object:Coxsackievirus B (CVB), which is divided into six serotypes(CVB1-6), 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 affords an opportunity to establish a new preventive procedure against CVB infections. VP1 is the major capsid protein of CVB3, and contains several T-and B-cell epitopes which can induce protective neutralizing antibodies. The gene encoding VP1 protein is the most promising candidate of CVB3 DNA vaccine. Some studies have shown that VP1 gene vaccine could effectively protect mice against CVB3 challenge. But the neutralizing antibody titers were low in these studies. The most probable reason is that VP1 protein can be expressed within transfected cells but can not release from these cells by itself and thus can not elicit antibody response effectively. Signal peptides may help proteinstransport out of cells, so fusing VP1 cDNA to signal peptide gene can probably promote secretion of VP1 protein. Human IL-2(hIL-2) signal peptide is a quite hydrophobic sequence containing 20 amino acids. It is cleaved off in the secretion process of mature hIL-2. Such cleavage could occur between Ser and Ala at positions 20 and 21. Cleavage site of signal sequence by signal peptidase is influenced by length of the hydrophobic region and secondary structure formation down-stream of the cleavage site. The object of this study was to construct a secretable VP1 eukaryotic expressing plasmid pcDNA3/sVP1 which express CVB3 VP1 by fusing it to the signal DNA sequence of the hIL-2 and 11 N-terminal amino acid residues of mature hIL-2. The functional immune response was evaluated by determining specific neutralizing antibody activity and by observing the survival condition of the mice after challenged with a lethal dose of the virus.Methods:Genes were spliced by overlap extension. Four primers were designed and synthesized. Primer 1 was 5′terminal sense sequence of hIL-2 signal peptide with a HindⅢ site. Primer 2 was 3′terminal antisense sequence of hIL-2 signal peptide with 18 added VP1 amino acid sequence. Primer 3 was 5′terminal sense sequence of VP1. Primer 4 was 3′terminal antisense sequence of VP1 with a XhoⅠsite. Signal peptide cDNA of hIL-2 wasamplified by PCR with primer 1 and primer 2 for primers and with pcDNA3/hIL-2 for template. CVB3 RNA was extracted from the culture supernatant of CVB3 (Nancy). The first strand of CVB3 cDNA was produced by reverse transcription with primer 4 for primer. PCR was performed with signal peptide cDNA of hIL-2 and the first strand of CVB3 cDNA for templates and with primer 1 and primer 4 for primers. The product of PCR was named sVP1. sVP1 cDNA was cloned into pGEM-T vectors and was verified by DNA sequencing. pGEM-T/sVP1 was digested with HindⅢ and XhoⅠ, and sVP1 cDNA was subcloned into eukaryotic expressing vector pcDNA3. The product was named pcDNA3/sVP1. pcDNA3/sVP1 was extracted from transformed E.coli DH5αby alkaline SDS lysis and was purified with PEG8000. BALB/c mice were inoculated intramuscularly (i.m.) in quadriceps muscle at 2-week intervals. Thirteen days after every injection , sera were collected and analysed for the titers of CVB3-specific neutralizing antibodies. Two weeks after the third immunization, mice were subjected to intraperitoneal (i.p.) challenge with 1000TCID50 CVB3 and the number of surviving animals was monitored up to 3 weeks post infection.Results:(1) A secretable VP1 eukaryotic expressing plasmid pcDNA3/sVP1 was constructed. DNA sequencing showed that the sequence of the sVP1 cDNA inserted wasidentical with that reported for VP1 cDNA, hIL-2 signal sequence and 11 amino acids. (2) The mean neutralizing antibody titers after every immunization in pcDNA3/sVP1 group were 1:9.33, 1:24.62, 1:27.32, respectively, and that in pcDNA3/VP1 group were 1:7.85, 1:9.01, 1:9.66, respectively. The mean neutralizing antibody titers in pcDNA3 group and...