All plasmids were prepared and purified using Endofree Mega-Q kits (Qiagen) for transfection and immunization. advantages such as simplicity of manufacture, high purity of product and ease of storage, DNA immunization has become an important direction of ERCC3 vaccine research and development. DNA vaccines against BoNTs induce protective humoral immune responses in mouse model, but when compared with conventional vaccines such as toxoid and subunit protein vaccines, DNA vaccines usually induce lower antibody level and protective efficacy and are still necessary to improve their potency for human use. A number of strategies have been investigated to increase the immunogenicity of DNA vaccines over the last few years, ranging from adjuvants, electroporation, cytokines, chemokines, CpG, viral replicon vector, liposomes to microparticles.17-21 In previous study, we indicated that plasmid DNA replicon vaccines encoding the Hc domains of BoNTs provide moderately efficient protection against BoNTs and more efficient potency than conventional plasmid DNA vaccines in mice.11,12 In our recently continual efforts to further refine and enhance the protective immune response of this antigen, we have proved that formulation of the DNA vaccines with aluminum phosphate adjuvant can efficiently enhance antibody responses and protective efficacy against BoNTs and GM-CSF gene adjuvant can augment the immunogenicity of JQEZ5 DNA replicon vaccine of BoNT/A.22,23 In the present study, we also evaluated the potency of IL-4 as a molecular adjuvant of DNA vaccines to enhance antibody responses and protective efficacy against BoNT/A in both Balb/c and C57/BL6 mice. Results Co-delivery IL-4 molecular adjuvant induces stronger humoral and protective JQEZ5 immune responses than DNA vaccine alone in mice To evaluate whether the immunogenicity of DNA vaccines could be increased by IL-4 molecular adjuvant, the humoral immune responses and protective effects of pVAX1AHc or pSCARSAHc by co-delivery pVAX1-IL-4 DNA vector were compared with the same DNA vaccines alone. As shown in Figure?1, the mean antibody titers to AHc in the vaccinated Balb/c mice with IL-4 were higher than those obtained from the vaccinated mice without IL-4 (p 0.05). The both DNA vaccines in the presence of IL-4 still predominantly induce Th2-type humoral immune responses as the both DNA vaccines alone, but with an IgG1 to IgG2a of ratio from approximately 10 to 26 for conventional pVAX1 DNA vector and 3 to 7 for replicon pSCAR DNA vector (Fig.?1B). These results suggest that stronger Th2-type humoral immune responses were modulated and elicited by the IL-4 molecular JQEZ5 adjuvant. Open in a separate window Figure?1. AHc-specific antibody responses in mice after i.m. vaccination with DNA vaccines. (A) Sera from each group of JQEZ5 mice at 4 weeks after the last immunization were collected, and the specific anti-AHc total IgG titers and individual IgGs isotype (IgG1 and IgG2a) titers were analyzed by ELISA. Serum samples from individual mice were assayed and the group mean titer (GMT) was calculated for the group (n = 8). (B) The IgG1/IgG2 ratio of serum antibodies was determined to evaluate the type of response to immunization. Results for each group represent the average ratio SEM *p 0.05; **p 0.01. Mice vaccinated with pVAX1AHc or pSCARSAHc co-delivered with pVAX1-IL-4 were completely protected against 1,000 50% lethal dose (LD50) of BoNT/A and partly protected against 10,000 LD50 of BoNT/A, while pVAX1AHc JQEZ5 or pSCARSAHc alone only provided part protection against 1,000 LD50 of BoNT/A and no protection against 10,000 LD50 of BoNT/A (Table 1). No protection was observed against 1,000 and 10,000 LD50 of BoNT/A in the negative control mice vaccinated with pVAX1-IL-4 or pVAX1. As shown in Table 1, the higher titers of neutralizing antibodies (0.32 or 0.64 IU/ml) were observed in the sera of mice vaccinated.