C3d Enhances Immune Response To The Secreted Form Of CVB3 VP1 DNA Vaccine

Object: Coxsackievirus group B(CVB), a member of the picornavirus group, is an important human pathogen. Among the six serotypes of CVB, CVB3 is increasingly recognized to be closely associated with acute/chronic myocardial disease and dilated cardiomyopathy cases. DNA vaccine is known for its ability to induce both humoral and cellular immune responses when delivered into animals. Its potential to generate specific CTL affords it the opportunity to establish new preventive procedures against viral infection. VP1, the major capsid protein of CVB3, can induce the production of effective neutralizing antibody. Although the vaccine constructed with VP1 alone could induce the production of antibody, the titers of antibody were usually too low to protect the host from lethal CVB3 challenge. Therefore, it appears to be an important strategy in our study to improve immunological potency and induce CVB3-specific immune responses, as well as enhance the protective efficacy.The complement system plays an important role in innate immunity. More recently, there has been an accumulating body of evidence indicating that the fragment of complement also participates in the adaptive immune response. C3d is one of the final degradation products of the third complement protein 3, which can't be hydrolysed any more by enzyme. C3d receptor, complement receptor type II (CR2, CD21), is primarily presented on the membranes of mature B cells and follicular dendritic cells (FDCs). When C3d is bond to foreign antigens (Ags), it can simultaneously bind to CR2+ cells. The attachment of foreign Ag to CR2+ cells through C3d facilitates the cross-linking of the Ag to the B cell membrane-bound receptor, which can lower the threshold of activating B cells, promote the affinity maturation of antibody and regulate the interaction of B cells and T cells. In this study, a fusion gene vaccine conjugating C3d3 to a secreted form of CVB3 VP1 was constructed. The immune effect was then evaluated by specific immune response and protection of mice with DNA vaccinations.Methods: (1) C3d3 cDNA was obtained by cutting the presented plasmid pSG5.C3d3.YL with proper restriction endonucleases. (2) The secreted form of CVB3 VP1 (sVP1) cDNA was amplified by PCR and cloned into pGEM-T vector. pGEM-T/sVP1 was then transformed E.coli DH5αand cultured in medium containing Ampicilin (100μg/ml) 2YT. The plasmids were extracted and identified by restriction endonuleases digestion as well as sequencing. (3) sVP1 was obtained from pGEM-T/sVP1 by proper endonucleases digestion and then inserted into pcDNA3 vector which was cut by the same endonucleases. As a result, a eukaryotic expressing plasmid of pcDNA3/sVP1 was formed. (4) Three copies of C3d were introduced into pcDNA3/sVP1 to construct pcDNA3/sVP1-C3d3 and restriction endonuleases digestion was used to identify the fused insertion. (5) Large preparation were performed to obtain the plasmids of pcDNA3/sVP1 pcDNA3/sVP1-C3d3 and pcDNA3 from transformed E.coli DH5α, and purified by PEG. (6) BALB/c mice aged 6-8 weeks were inoculated intramuscularly (i.m.) three times at four weeks'interval with 100μg of pcDNA3/sVP1-C3d3, pcDNA3/sVP1 and pcDNA3 plasmids respectively. Fourteen days after every injection, sera of each group were collected and the titers for CVB3-specific neutralizing antibodies were measured. Three weeks after the third immunization, splenocytes from three immunized mice of each group were stimulated by inactivated CVB3 and harvested to analyze the lymphocytic proliferative activity and specific CTL cytotoxic activity by CCK-8 assay. At the same time, another eight mice were challenged with 5LD50 CVB3 and the number of surviving animals was monitored up to three weeks post infection. Furthermore, the rest mice of each group were challenged with 3LD50 CVB3 and sacrificed after seven days in order to evaluate the titers of blood viruses. Meanwhile, hearts of mice was fixed with 10% formalin, dehydrated with grading ethanol, embedded in paraffin, and cut into 5μm sections. Some sections were stained with hemotoxylin and eosin to observe the changes of the myocardium. Results: (1) Gene fragment of C3d3 was obtained by restriction endonucleases digestion and sVP1 fragment was amplified by PCR. After restriction endonucleases digestion, both of the length was the same as expected. DNA sequencing showed that sVP1 was identical to the sequence recorded in Genbank. (2) Three copies of C3d were introduced into pcDNA3/sVP1 to form pcDNA3/sVP1-C3d3. The result of enzyme digestion was identical as expected and suggested that the eukaryotic expressing plasmid pcDNA3/sVP1-C3d3 had been constructed successfully. (3) When mice were immunized with the plasmids, the antibody titers increased with the time of inoculation. The mean titers of neutralizing antibody after every immunization in pcDNA3/sVP1 group were 1:8.91,1:15.87,1:28.28, respectively, and that in pcDNA3/sVP1-C3d3 group were 1:8.41,1:25.19,1:33.65, while it is always lower than 1:5 in mice immunized with pcDNA3. More specifically, mice with pcDNA3/sVP1-C3d3 elicited the strongest neutralizing antibody after the third immunization. When compared with that of pcDNA3/sVP1, the difference was significant (P<0.05). (4) Splenocytes from vaccinated mice showed different proliferative activity due to different stimulants of CVB3 or ConA. When stimulated by CVB3, the splenocytes from mice with pcDNA3/sVP1-C3d3 showed significantly stronger proliferative activity than that with pcDNA3/sVP1 (P<0.05). While when stimulated by ConA, the proliferative activity of mice with pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 were both stronger than that with pcDNA3, but the difference between the two stronger groups was not significant. (5) DNA expressing sVP1 fused to three copies of C3d efficiently enhanced the specific lymphocytic CTL cytotoxic activity, which was remarkably stronger than that in the mice immunized with pcDNA3/sVP1. (6) Protection of mice from death after lethal CVB3 (5LD50) challenge was augmented to 50% with fused DNA, while mice with pcDNA3/sVP1 was only 25% and no one survived in pcDNA3 group. Chi-square test indicated that differences between any two groups were not significant by the multiple comparisons. Kaplan-Meier test was used to compare the differences of survival curves, which indicated that the survive state of pcDNA3/sVP1-C3d3 group is better than that of pcDNA3 group (P<0.05), but difference between pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 was not significant (P>0.05). (7) After 3LD50 CVB3 challenge, the virus titers of blood in pcDNA3/sVP1-C3d3 group were only 2.58±0.66, significantly lower than that of pcDNA3/sVP1 group (3.61±0.41). (8) The histopathology changes in mice immunized with fused DNA were less serious compared with that in mice of other groups, but significant difference couldn't be found between them.Conclusion: (1) The gene fragment of C3d3 was obtained by restriction endonucleases digestion successfully. (2) The eukaryotic expressing plasmid pcDNA3/sVP1-C3d3 was constructed successfully. (3) The survival rate of mice with pcDNA3/sVP1-C3d3 peaked to 50%, and pcDNA3/sVP1 was 25%, while no mice survived in pcDNA3 group. The differences were not significant by Chi-square test. While, the result of Kaplan-Meier test showed that the survival state of pcDNA3/sVP1-C3d3 group was better than that of pcDNA3 group (P<0.05). (4) After administration of pcDNA3/sVP1-C3d3 and pcDNA3/sVP1 to BALB/c mice, the neutralizing antibody increased along with the inoculation times. More meaningfully, C3d3 could induce much stronger antibody from mice. (5) Specific lymphocytic proliferative activity and CTL cytotoxic activity in immunized mice with pcDNA3/sVP1-C3d3 were markedly stronger than those in mice immunized with pcDNA3/sVP1 and pcDNA3. (6) Overall, the results of neutralizing antibody titers, specific lymphocytic proliferative activity, specific CTL cytotoxic activity, viruses titers and histopathology all indicate that C3d can strongly enhance the specific immune response induced by sVP1 gene vaccination, but the protection of mice from death after lethal CVB3 (5LD50) challenge are still not significant now.