A Novel Strategy To Enhance Immune Responses Of Canine Hepatitis DNA Vaccine By Coadministration With PVAX1-CpG-Loop And Adjuvant

Objective Nucleic acid vaccine is a new vaccine technique following inactivated vaccines and attenuated vaccines in 1990s. Nucleic acid vaccine is capable of eliciting both cellular and humoral immune responses more complete than conventional vaccination and have the advantage of mass-production easily. It has been actively developed in the past few years against a variety of infectious agents including virus, bacteria and parasites. Beause of it's small antigen molecule,higher level of purification, short of immunogenicity,the effective adjuvant was needed to strengthen the immune response that induced by DNA vaccine. Efficient adjuvant is beneficial for DNA vaccine to enhance sufficient level of protection and long-lasting immunity.Synthetic oligodeoxynucleotides (ODNs) that contain immunostimulatory CpG motifs trigger an immunomodulatory cascade that involves B and T cells, natural killer cells and professional antigen-presenting cells. The response to CpG-ODNs skews the host's immune milieu in favour of T helper 1 (Th1)-cell responses. In mice, immune cells of the myeloid lineage including monocytes, macrophages and myeloid DCs express TLR9 and respond to CpG stimulation.The cationic surfactant dimethyl dioctadecyl ammonium bromide (DDA) is a novel adjuvant, DDA can adsorb most antigens on their surface and promote a strong cell mediated immune response and a humoral immune response in vivo, which is essential for the induction of protective immunity against most diseases.Lipofectamine TM 2000 is a cationic lipid, liposome has been widely used to enhance DNA vaccines and preferentially activate Th1 responses. in vitro, the lipide and DNA compounds can fuse with the celluar membrance and help the DNA come into the cytolymph .In previous studies, we have constructed the DNA vaccine (pVAX1-CpG-Loop) that encoding two major Loop DNA fragments from CAV-1 and it has induced significantly cell mediated and humoral immune responses. In order to get the better immune efficacy in mice, especially induce a more durable T-cell response, we compared the effect of the three adjuvants on pVAX1-CpG-Loop DNA vaccine. This study evaluated the adjuvant effects of CpG-ODN, DDA, and LipofectamineTM 2000 on a DNA vaccine pVAX1-CpG-Loop, which was against canine adenovirus type-1. Selecting the appropriate adjuvant to specific antigenic components will not only enhance the level of immune response but also determine the type of immune response induced.Methods 1 Plasmid isolation and purificationThe plasmid pVAX1-CpG-Loop was isolated by the method of alkaline lysis, and purified by polyethylene glycol precipitation. The fusion protein was purified by Ni-NTA affinity chromatography column.2 preparation of adjuvantsThe CpG-ODN (1826) sequence is 5'-TCCATGACGTTCCTGACGTT-3', which was produced with DNase-protected phosphorothioate bonds backbone. CpG-ODN were dissolved in a sterile phosphate buffered saline solution and stored at -20℃.DDA adjuvant was prepared as described previously. Briefly, DDA adjuvant was mixed into 10 mM Tris-buffer at pH 7.4 to a concentration of 1.25 mg/ml, heated to 80℃for 20 min with intermittent shaking, then cooled to room temperature.3 MiceBALB/c mice were bred and cared in the Animal Facilities of Hebei Medical University, Shijiazhuang. Only female mice ages 6-8 weeks old were used for the experiments.4 Immunization protocolsExperiment 1: female mice were randomly divided into 6 experimental groups (5 mice /group). The detailed vaccination scheme were as followed. Biweekly mice were injected intramuscularly with a volume of 100μl/mouse for three times. After two weeks, the splenocytes (5 mice / group) were harvested. The Intracellular staining of IFN-γ,In vitro CTL activity assay and The lymphocyte proliferation assay was performed.Group1. pVAX1-CpG-Loop 25μg/mouseGroup2.pVAX1-CpG-Loop25μg/mouse+CpG-ODN50μg/mous-eGroup3.pVAX1-CpG-Loop25μg/mouse+CpG-ODN20μg/mous-eGroup4.pVAX1-CpG-Loop25μg/mouse+LipofectamineTM 2000 (Invitrogen USA)40μl/mouseGroup5.pVAX1-CpG-Loop 25μg/ mouse +DDA 250μg/mouseGroup6.pVAX1-CpG-Loop 25μg/ mouse +DDA 25μg/mouseExperiment 2: The grouping project same as Experiment 1. Biweekly mice were injected intramuscularly with a volume of 100μl/mouse for three times. All the groups were re-injected with pVAX1-CpG-Loop (25μg/mouse) at thirteenth, fifteenth and eighteenth week respectively. two weeks later after the final immunization, the splenocytes were harvested. The Intracellular staining of IFN-γ, In vitro CTL activity assay and The lymphocyte proliferation assay was performed.Experiment 3: The mice were injected intramuscularly (100μg/100μl/mouse, 5 mice /group) with the DNA vaccine and the adjuvants at a 2-week interval for three times. After two weeks, the splenocytes were harvested. The Intracellular staining of IFN-γ, In vitro CTL activity assay and The lymphocyte proliferation assay was performed.Group1.pVAX1-CpG-Loop100μg/mouse+CpG-ODN50μg/mo- useGroup2.pVAX1-CpG-Loop100μg/mouse+CpG-ODN20μg/mo-useGroup3.pVAX1-CpG-Loop 100μg/ mouse + LipofectamineTM 2000 40μl/ mouseGroup4.pVAX1-CpG-Loop100μg/ mouse +DDA250μg/ mouseGroup5. pVAX1-CpG-Loop 100μg/ mouse5 The evaluation of the immuned mice5.1 Enzyme-linked immunosorbent assaySera of immunized mice were collected and specific IgG antibody was detected by the method of indirect enzyme linked immunosorbent assay (ELISA) as described previously . Briefly, the dilution of serum sample was 1:40, purifed CAV-1 was used as antigen and specific anti-virus IgG antibody was detected. Absorbance was determined at 492 nm.5.2 The Loop protein specific lymphocyte proliferation assayOD 570 was measured by a standard 3-(4,5dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) method. The stimulation index was calculated from the formula: stimulation index (SI) = (Arestimulated -Ablank)/(Aunrestimulated-Ablank)5.3 Intracellular staining of IFN-γ1×107 spleen cells from immunized mice were cultured in 0.5 ml of complete DMEM in the presence of the recombinant Loop protein 30μg/ml and 10 ng/ml of interleukin-2 for 72h. The stimulated lymphocytes were harvested and labeled for surface CD8 and intracellular IFN-γusing PE (Jingmei Biotech) or FITC (BioLegend) labeled specific monoclonal antibodies following the detailed instructions provided by the vendor. Spleen cells were analyzed by flow cytometry.5.4 The Loop protein specific CTL activity assay5.4.1 preparation of the effectors :The cell line CT26 was transfected with plasmid pVAX1-CpG-Loop or pVAX1-CpG vector with Lipofectamine 2000 according to the manufacturer's direction as the method described previously. After 48 h incubation at 37℃, 5% CO2, CT26 cells were collected and the transfection products were analysed by RT-PCR and Western blotting respectively.5.4.2 Preparation of the target cells: splenocytes from primed mice (1×107 /well) were cultured with the recombinant Loop protein (30μg/ml) and recombinant mouse IL-2 (10 ng/ml) in DMEM medium for 7days in 6 -well tissue culture plates.5.4.3 CTL activity assay: The restimulated splenocytes were effectors. Transfectant CT26 cells (transfected with plasmid pVAX1-CpG-Loop) were used as target cells. The mean percentage of specific lysis in triplicate wells was calculated as follows: % specific lysis = [(experimental release-spontaneous release)/(maximum release-spontaneous release)]×100.6 Statistical analysis Unless noted, Comparisons between two groups were performed by two-tailed Student's t-test. A p-value < 0.05 was considered as significant.Results1 The recombinant plasmid pVAX1-CpG-Loop extracted abundantly by alkaline lysis was identified by double enzyme digestion. The electrophoretic band about 860 bp could be observe under ultraviolet light about 1.2﹪agarose gel electrophoresis. The purity and concentration of the extracted recombinant plasmid detected by grating spectrophotometer demenstrated that the ratio of OD260/OD280 was between 1.8 and 2.0. Western blot analysis indicated that the recombinant protein could be recognized by His-specific McAb; The purity of Loop protein was about 95% after purification with Ni-NTA affinity chromatography column;2 Higher levels of Ag-specific IgG were induced by coimmunization with adjuvants and DNA vaccine. In the experiment2 and 3, in contrast to the pVAX1-CpG-Loop (25 or 100μg/ mouse) group, higher levels of IgG were elicited in DNA vaccine plus adjuvants groups (all p<0.05). However, no significant differences of the levels of IgG in DNA vaccine plus different adjuvants were observed (p>0.05).3 Splenocytes from immunized mice with pVAX1-CpG-Loop plus adjuvants showed markedly higher proliferative response than those from immunized with pVAX1-CpG-Loop DNA vaccine solely in experiment 2 and in experiment 3 (all p<0.05) . However, the mice showed no porliferative response in experiment 1(all p>0.05) (Fig.2C).4 11-29% of the splenic CD8+ T cells from mice that in the experiment 2 produced IFN-γ. There were significant differences between control and coimmunization groups, all p < 0.05. Whereas only 1.86-2.87% of the splenic CD8+ T cells from mice vaccinated with the DNA vaccine plus adjuvant produced IFN-γin the experiment 3. Compared with control, all p < 0.05. But only 0.26-0.58℅ of the spleen CD8+ T cells from mice in the experiment 1 produced IFN-γ, all p < 0.05.5 Significantly higher CTL responses to the Loop protein were generated in mice in the experiment 2. In experiment 3, the mice vaccinated with a higher dose (100μg) of DNA vaccine plus adjuvants induced weaker CTL responses than that in mice in the experiment 2. Significantly lower CTL responses were generated in mice in the experiment 1 than that in the experiment 3. These results further revealed that the lower dose (25μg/ mouse) of DDA plus pVAX1-CpG-Loop DNA vaccine generated significantly higher CTL response than the higher dose (250μg/ mouse) of DDA coinjecting DNA vaccine in mice in the experiment 2.Conclusions:1 The higher level of IgG was elicited by the lower dose of DNA vaccine and the adjuvants in experiment 2. when the vaccination frequency was added, our data indicated that the adjuvants had higher efficency in humoral immune responses even on immunization with the lower dose of pVAX1-CpG-Loop.2 The DNA vaccine plus adjuvant eliciting significantly higher level of T-cell proliferation and CTL response than the control group in experiment2 and 3. The data indicated that higher frequency of CD8+ T cells and higher level IFN-γproduction appeared in experiment2 and 3.3 These studies showed that DDA, CpG-ODN and LipofectamineTM 2000 had corresponding adjuvant effect and the augmented effect on DNA vaccine with CpG motif were observed. The strategy that priming with the lower dose DNA vaccine coadministered with adjuvant and boosting with the lower dose DNA vaccine only is the most potent vaccination regimen known to date for DNA vaccine (pVAX1-CpG-Loop) to induce antiviral response.