Construction Of Fusion Gene DNA Vaccine PcDNA3/MDC-VP1 And Study On Its Immunological Effect On Mice

Objective: Coxsackievirus B3(CVB3) infections arecommon causes of acute and chronic myocarditis in human.Despite the well charactered molecular structure of Coxsackieviruses, no virus-specific preventive procedures against CVB3are clinically available today. Immunization with DNA vaccinemakes it possible to establish a new preventive procedureagainst CVB infections. VP1 is a major capsid protein of CVB3,which can induce the production of effective neutralizingantibody. Although the vaccine constructed with VP1 alonecould induce the production of antibody, the titers of antibodyproduced were usually too low to protect the host from lethalCVB3 challenge. Therefore, improving the immunologicalpotency ,particularly the Ab response, has been a seriouse hurdleto enhancing the protective efficacy of DNA vaccine encondingvp1. Antigen-presenting cells (APCs) play a central role inpriming immune responses . Targeting strategies,which targetAg to APCs, are being developed to enhance theimmunogenicity of DNA vaccines. Ags linked to molecules suchas CTLA-4 and L-selectin have been used to targete Ags toAPCs , and have been proved that the immune responses wreenhanced in different species with some degrees of success.Macrophage-derived chemokine (MDC, CCL22) was firstisolated by random sequencing of cDNA clones from humanmonocyte-derieved macrophages and showed chemotacticactivity of monocytes, macrophages, dendritic cells, and IL-2activated natural killer cells. Similar chemokines were alsoisolated from human activated microphages, and murine B cellsand dendritic cells,and were termed stimulated T-cellchemotactic protein (STCP)-1 and ABCD-1, respectively. MDCis a member of the CC chemokine family , and has been shownto bind to CC receptor 4 (CCR4), which are mainly expressedon Th2 cells, B cells, Macrophages, et al. Although dendriticcells and monocytes, are responsive to MDC, they do notexpress CCR4. It han been suggested that MDC may recognizeanother, yet unidentified receptor, such as CCR4 variant (GarciaGE, 2003). MDC ia a strong chemotactic agent to T cells,especially Th2 cells , plays an important role for the traffickingof Th2 cells and the induction of Th2-related immune responses.Therefore , we postulated that fusing MDC to VP1 couldenhance the effect of CVB3 vaccines. In this study, MDC genewas linked to CVB3 VP1 gene to construct an eukaryoticexpressing plasmid pcDNA3/MDC-VP1, based on an idea thatthe fusion protein MDC-VP1 would be expressed in the animalcells after inoculation and targeted VP1 to APC by the MDCprotein recognizing MDC receptors on the cell surface of APCs.To maintain the natural spatial stuctions of MDC protein andVP1 protein, a DNA sequence which encode a flexiblepolypeptide (Linker) was used.Methods: (1) The total RNA of spleen cells from scaldmice, which have been stimulated with LPS for 24 hours, wasextracted. The DNA fragments of MDC and MDC-Linker(MDC-L) was amplified by RT-PCR from the total RNA ofspleen cells. Similarly, the DNA fragments of VP1 andLinker-VP1(L-VP1) was amplified by RT-PCR from the RNAof CVB3. (2) The PCR products of the four DNA fragments waslinked to plasmid pGEM-T , E.coli DH5αwas transformed andcultured in medium containing Ampicilin (100 ug/ml) 2YT.Then plasmids were extracted, recombinant clones wereselected and identified by endonuclease cutting and sequencing.(3) The constructed plasmid were cut by proper endonucleasesand the target gene fragments were linked to pcDNA3 whichwas cut by the same endonucleases to construct the eukaryoticexpressing plasmids of pcDNA3/MDC , pcDNA3/VP1, andpcDNA3/ MDC-VP1. (4)Large preparation were performed toobtain the plasmids of pcDNA3/MDC , pcDNA3/VP1, andpcDNA3/ MDC-VP1 and pcDNA3 from transformed E.coliDH5α, and purified by PEG. (5) BALB/c mice aged 6-8 weekswere divided into 6 groups at random : Saline control group,pcDNA3 control group, pcDNA3/MDC group, pcDNA3/VP1group, pcDNA3/VP1 and pcDNA3 /MDC group, andpcDNA3/MDC-VP1 group. Every group consisted of 26 mice.The plasmid were inoculated to mice in a dose of 100ug ofDNA/mouse, intramuscularly (i.m.) in quadriceps muscle at3-week intervals. Twenty days after every injection , sera werecollected and CVB3-specific neutralizing antibodies weretitrated. Three weeks after the third immunization, 20 mice fromeach group were subjected to intraperitoneal (i.p.) challengewith 10 LD50 of CVB3 and the number of surviving animalswas monitored up to 3 weeks post infection. The rest mice ofeach group challenge with 3 LD50 CVB3 and were sacrificed at7th day, the titers of blood viruses and the ratio of heatweight versus body weight (HW/BW) were evaluated. Then thehearts fixed with 10% formalin ,dehydrated with gradingethanol, embedded in paraffin, and cut into 5 μm sections.Some sections were stained with hemotoxylin and eosin toobserve the changes of the myocardium.Results: (1) The gene fragments of MDC, MDC-L, VP1and L-VP1 was amplified by RT-PCR. The length of thefragments was same as expected.(2)The prokaryotic clonevectors pGEM-T/MDC, pGEM-T/MDC-L, pGEM-T/VP1 andpGEM-T/L-VP1 were successfully constructed , DNAsequencing showed that the sequences is identical to thesequence recorded in Genbank.(3) After these gene fragmentswere inserted to the pcDNA3, the recombined plasmid wasidentified by enzyme cutting and the results were identical asexpected and suggested that the eukaryotic expressing plasmidof pcDNA3/MDC , pcDNA3/ VP1 and pcDNA3/ MDC-VP1had been constructed successfully. (4) The mean titers ofneutralizing antibody after every immunization in pcDNA3/VP1group were 1:5.95, 1: 9.01, 1: 16.82, respectively, and that inpcDNA3/VP1+ pcDNA3/MDC group were 1: 6.37, 1: 10.72, 1:26.39, respectively, and that in pcDNA3/MDC-VP1 group were1: 6.83, 1: 18.03, 1: 33.64 ,respectively. The mean neutralizingantibody titers in pcDNA3 group , N.S. and pcDNA3/MDCgroup were all lower than 1:5. The statistics analysis showedthat the diffrences of neutralizing antibody titers of every groupproduced after three times of inoculation were significant; Afterthe third inoculation, the mean neutralizing antibody titers inpcDNA3/MDC-VP1 group and pcDNA3/VP1+pcDNA3/MDCgroup were higher than pcDNA3/VP1. The difference weresignificant when analysed by the multiple comparison (P<0.05).But the difference between pcDNA3/MDC-VP1 group andpcDNA3/VP1+ pcDNA3/MDC group was not significant. (5)Up to the 21th day after CVB3 challenge, the survival rates ofpcDNA3/MDC-VP1 group, pcDNA3/VP1+ pcDNA3/MDCgroup, pcDNA3/VP1 group, pcDNA3/MDC group, pcDNA3group and N.S. group were 50%, 35%, 25%, 20%, 10% and15%, respectively. Chi-square test indicated that there weredifferences among groups(P=0.048), but differences beteen anytwo groups wre not significant by the multiple comparison.Kaplan-Meier test was used to compare differences of survivalcurves, the result showed that the difference was significant(P<0.05). The result of multiple comparison indicated that thesurvive state of pcDNA3/MDC-VP1 group is better than that ofpcDNA3/VP1 group (P<0.05), but difference beteenpcDNA3/VP1 + pcDNA3/MDC group and pcDNA3/VP1 wasnot significant (P>0.05). (6)The virus titers of blood inpcDNA3/MDC-VP1 group and pcDNA3/vp1 + pcDNA3/MDCgroup were lower than that of any other group, the differencewas significant (P ≤0.05), but difference betweenpcDNA3/MDC-VP1 group and pcDNA3/VP1 + pcDNA3/MDCgroup was not significant. (7) HW/BW in all group, exceptpcDNA3 group, are smaller than N.S. group, the multiplecomparison indicate the differences were significant (P<0.05).HW/BW in pcDNA3/MDC-VP1 group and pcDNA3/VP1+pcDNA3/MDC group were smaller than other groups (P<0.05).(8) The histopathology changes in pcDNA3/MDC-VP1 groupand pcDNA3/vp1 + pcDNA3/MDC group were less seriouscompared with that of the other groups, but no significantdifference were found between them. (9) Differences in micebody weight between 0th day (just before inoculation) and 63thday (just before challeng with CVB3) were analysed. Thedifferences were not significant.Conclusion: (1) The gene fragments of MDC and CVB3VP1 was succesfully cloned by RT-PCR. (2) The eukaryoticexpressing plasmid pcDNA3/MDC, pcDNA3/MDC-VP1 andpcDNA3/VP1 was constructed successfully; (3) The survivalrates of pcDNA3/MDC-VP1 group, pcDNA3/VP1+pcDNA3/MDC group, pcDNA3/VP1 group were 50%, 35%,25%, respectively, the differences were not significant. The...