【作者】 任继玲；

【导师】 于永利；

【作者基本信息】 吉林大学 ， 免疫学， 2010， 博士

【摘要】 CpG ODN是含有非甲基化胞嘧啶—鸟嘌呤二核苷酸的单链脱氧寡核苷酸,通过与TLR9结合,能刺激人及哺乳动物的天然免疫系统,诱导产生以Th1型为主的免疫应答。CpG ODN作为人用疫苗佐剂在临床实验中取得了可喜的成果,作为动物疫苗佐剂的研究较少,这是因为CpG ODN具有种属特异性,即对一种动物表现生物活性的CpG ODN,在另一种动物则未必表现出同样的或同样强度的生物学活性。实验证明以CpG为核心的六核苷酸即CpG基序是导致种属特异性的原因。为了获得对牛或犬有免疫刺激作用的CpG ODN,以作为动物疫苗新型佐剂,我们根据CpG基序设计了60多条含有各种CpG基序的CpG ODN,经过体外筛选,获得了对犬免疫细胞有刺激活性的YW07、YW08和YW09；将YW07与含有铝佐剂的狂犬灭活疫苗联用免疫小鼠及犬,YW07能显著提高抗体产生水平及持续时间,并偏向Th1型免疫应答,且完全保护小鼠抵抗狂犬病毒的攻击。同时,获得了对牛免疫细胞有刺激活性的RW12,将RW12与含有206油佐剂的口蹄疫重组蛋白A7疫苗联用免疫小鼠及牛,RW12能显著提高其在动物体内的抗体水平及持续时间,一次免疫84天后进行攻毒实验,A7+206油+RW12组的牛80%(4／5)得到保护,灭活疫苗组只有50%(2／4)的牛得到保护。综上所述,我们开发的牛、犬特异性CpG ODN有望成为动物疫苗的新型高效佐剂。更多还原

【Abstract】 Rabies is a viral disease that may affect human central nervous system and cause a fatal neuroencephalomyelitis. Every year,40,000-70,000 people die of rabies worldwide. The domestic dogs are the main reservoirs and vectors for spreading rabies virus and account for more than 99% of all human rabies cases. Obviously, it is an urgent requirement to promote programmed mass vaccination of dogs in the countries with endemic canine rabies. Currently, a yearly prophylactic shot of inactivated alumimun-adjuvanted rabies vaccines (IARV) is being used to immunize dogs to control rabies. However, a single injection of the vaccines, as reported, failed to induce protective and long-lasting immune response in a portion of dogs. Routinely, Alum is included in the rabies vaccines to improve its efficacy. Although safe and well tolerated, the Alum is strongly Th2 biased and relatively weak as an adjuvant. Ideally, a rabies vaccine should be able to prime a Thl/Th2 balanced and efficient immune response to eliminate rabies virus infections. As reported, high titer of IgG2a is significantly correlated with the survival from lethal rabies virus challenge in mice. Apparently, it is required to develop a novel adjuvant combined with or substitute for alum, to promote the efficacy of current rabies vaccines of dog use.Foot-and-mouth disease (FMD) is a highly contagious disease of cloven-hoofed animals. Recent FMD outbreaks in some countries and their significant economic impact have increased the concern of governments worldwide. Inactivated FMDV vaccine is currently used to control the disease. The introduction of the killed FMD vaccine has been extremely successful in reducing the number of FMD outbreaks in many parts of the world. However, there are safety concerns. one is the possible escape of live virus from vaccine production. The other is the incomplete inactivation of the virus may cause the outbreak of FMD, as reported in Europe for several times. In addition, the duration of antibody induced by inactivated FMDV vaccine is usually short, needing a booster injection. In our lab, we have developed an effective recombinant FMDV vaccine, named A7. To enhance the immune effect of A7, we need to develop a novel adjuvant.In recent decade, multiple studies have shown that synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotide (CpG ODN) are potent adjuvants. After recognizing toll like receptor 9, the CpG ODN activates B cells, inhibits apoptosis of B cells, and induces the maturation and differentiation of dendritic cells. In mice, the CpG ODN facilitates Thl-biased immune response characterized by enhancing the secretion of IFN-y, TNF-a, IL-12 cytokines and IgG2a antibodies, and the generation of specific cytotoxic-T-lymphocyte (CTL). These properties have been stirring up the studies using CpG ODN to improve currently used vaccines. When used in combination with alum, CpG ODNs can greatly enhance antibody responses to hepatitis B surface antigen in non-human primates and humans. CpG ODN is species specificity. It has been known that CpG ODN containing varied CpG motifs may differentially stimulate various mammalian cells, and that CpG motifs, based on their species specificities, can be described as mouse (GACGTT), human (GTCGTT) and porcine (ATCGAT) motifs, respectively. However, potential applications of CpG ODN in species of veterinary are just being explored. We need to develop bovine or canine favored CpG ODN for adjuvant use.Based on CpG motif, we have tried to design a series of CpG ODNs containing different CpG motifs. Three AACGTT motif-containing CpG ODNs, designated as YW07, YW08 and YW09, respectively, stood out with vigorous capability of activating canine immune cells. The CpG ODNs were further studied for their adjuvant activity for rabies vaccines in mice and dogs. Meantime, RW12 was identified as bovine favored CpG ODNs, which was tested in mice or cattle as adjuvant for recombinant FMDV vaccine A7.The results of this paper including the following parts:1. Designing and screening candidate canine favored CpG ODNsWe successfully established an optimal condition for screening canine favored CpG ODNs, In 96 well round bottom plate,6×105 canine splenocytes/well stimulated by 10μg/ml CpG ODN for 48 hours can resulted high stimulation index by 3H-thymidine incorpration. To develop novel CpG ODNs of dog use, we have tried to design a series of CpG ODNs containing different CpG motifs, including GACGTT, GTCGTT, AACGTT, ATCGTC and ATCGAT. We noticed that AACGTT motif-containing CpG ODNs, such as YW07, YW08 and YW09 could consistently induce a strong proliferative response in canine splenocytes (P< 0.001 vs. medium). The activity increased sharply when the dose of YW07, YW08 and YW09 were increased from 2.5 to 10μg/ml, and reached a plateau level at 10μg/ml. It was showed that YW07, YW08 and YW09 could stimulate mouse splenocytes to proliferate vigorously as compared with medium control (P< 0.001). The activity was as potent as those of 1826 and 2006 (P> 0.05).2. Testing the adjuvant activity of CpG ODN for IARV in miceUpon the in vitro screening, YW07, YW08, YW09 were selected to test their adjuvant activity for canine rabies vaccine in mice. Mice in five groups (six mice each) were immunized with IARV or IARV plus YW07, YW08, YW09 or 2006, respectively. Mice in two additional groups received either 10μg YW07 or placebo respectively. On day 14 after the immunization, the level of anti-RV antibody induced by IARV+YW07 was significantly higher than those induced by IARV+YW08, IARV+2006 or IARV+YW09, or IARV respectively (P< 0.05). The peak anti-RV antibody level was reached on day 28. Noticeably, the peak antibody level induced by IARV plus YW07 was sustained for 112 days without apparent drop. The anti-RV antibody induced by IARV+YW07 was 1.5,1.6,1.9 or 2.8-fold higher than those induced by IARV+YW08, IARV+2006, IARV+YW09, or IARV alone on day 112 post-immunization.Anti-RV IgGl and IgG2a in the sera collected on day 28 were measured by ELISA. The IgGl/IgG2a ratio induced by IARV+YW07 was similar with that induced by IARV+ YW08 (P= 0.11) and significantly lower than those induced by IARV (P< 0.001), IARV+2006 (P< 0.05) or IARV+YW09 (P< 0.001), respectively. The data indicate that the addition of YW07 and YW08 to IARV resulted in a shift towards a Thl-biased immune response.To check the protective efficacy of the induced immune response, all the mice immunized with IARV plus various CpG ODNs were challenged intracerebrally with 50 LD50 of virulent rabies virus CVS strain on day 112. All of the mice injected with placebo or YW07 only failed to resist the challenge and died on day 5 to 10 after challenge. All of the mice immunized with IARV+YW07, IARV+YW08, IARV+ YW09, IARV+2006 resisted the challenge and survived, whereas half of the mice immunized with IARV only were protected. A dose analysis showed that YW07 could enhance IARV to induce high level anti-RV antibodies in a dose-dependent manner in mice. This result further confirmed that YW07 could enhance the immunogenicity of IARV even at a lower dose, such as 2.5μg per mouse. The IgGl/IgG2a ratio in mice inoculated with IARV plus YW07 at 2.5μg per mouse was significantly lower than that in the mice inoculated with IARV alone (P< 0.001). When mice inoculated with IARV plus YW07 at 10μg or 20μg, the IgG1/IgG2a ratios were significantly lower than those in all other groups (P< 0.001). Significantly, all the mice immunized with IARV plus YW07 even at a lower dose, such as 2.5μg per mouse, resisted the challenge and survived.We further investigated whether YW07 could show similar effect in dogs. Dogs in two groups were immunized with IARV only or IARV plus 20μg YW07, respectively. As early as 7 day after the immunization, the level of anti-RV antibody induced by IARV+20μg YW07 were significantly higher than that induced by IARV only (P< 0.05). Noticeably, on day 84, the anti-RV antibody induced by IARV+20μg YW07 was 2.3-fold higher than those induced by IARV alone (P< 0.01). The data show that YW07 could facilitate IARV to induce a vigorous, early produced and long lasting anti-RV antibody response in dogs, which implies that YW07 is a potent adjuvant for canine rabies vaccine to control rabies virus infection.3. Bovine specific CpG ODNs’designing and screeningIn 96 well round bottom plate,2×105 canine splenocytes/well stimulated by 10 mg L-l CpG ODN for 72 hours can resulted high stimulation index by 3H-thymidine incorpration. For developing novel bovine favored CpG ODNs, we choose 32 CpG ODNs from our lab pool, named RW01-RW32. RW12 could stimulate bovine PBMC or splenocytes to proliferate vigorously, the effect was higher than 2006, which indicated RW12 was specific for calltle. RW12 contains AACGTT and ATCGTC motifs. To evaluate how the CpG motif contributes to the activation of immune cells, we synthesized derivatives, which differ from RW12. When AACGTT was replaced by AAGCTT, RW12-3 induces 10% level of immune cells’proliferation than RW12. When ATCGTC was replaced by ATGCTC, the activity reduced to 45% of RW12. When ATCGTC was replaced by GTCGTT、AACGTT、ATCGA motif, the effect was similar with that induced by RW12-4, which just contains AACGTT motif. The data demonstrate AACGTT motif was important than ATCGTC motif, ATCGTC motif could assist AACGTT motif. RW12 could stimulate the proliferation of immune cells from human, mouse, guinea pig, rabbit, dog or pig, the effect was similar with 2006 (P> 0.05 vs.2006). The flow cytometry analysis showed RW12 up-regulated CD69 in CD 19+, CD56+, CD 14+, indicating that YW12 could activate human B cells and NK cells. RW12 could up-regulate CD80, CD86 and HLA-DR in human PBMC. All these results suggest RW12 can activate lymphoid cell activation.4. The enhance effect of RW12 for oil-based recombinant FMDV vaccine A7 in mice and cattleFMDV recombinant protein A7 was constructed by Xu haifei. The pET28a-RA2 was transformed into BL21. After being induced by IPTG, the bacteria was collected and lysed. The recombinant protein A7 was purified through nickel chromatography.Upon the in vitro screening, RW12 was selected to test its adjuvant activity for A7 in genuine pigs. There are five groups A7, A7+alum, A7+alum+RW12, A7+206 oil, and A7+RW12. The antibody level induced by A7 was significantly higher than that induced by other groups (P< 0.05). RW12 failed to assist A7 to produce higher antibody, which may be due to protein polymerization when meet nucleotides with negative charge.We further try various combinations of A7 and RW12 in mice to find the optimal effect. There are five groups A7+206 oil, A7+alum+206 oil, A7+206 oil+RW12, A7+alum+206 oil+RW12, and A7+alum. The antibody level induced by A7+206 oil+RW12 was significantly higher than that induced by other groups (P< 0.05). The high antibody level kept for 175 days without apparent drop. Noticeably, A7+206 oil +RW12 could induce more IgG2a than other groups.To check whether A7+206 oil+RW12 could induce efficient immune response in cattle, A7+206 oil, A7+alum+206 oil, inactivated virus vaccine and PBS was immunized in cattle. The serum were collected and tested by Elisa, virus neutralization using BHK-2 and suckling mice protection. The results show that high titer antibodies can be induced in cattle by A7. Antibodies can also be induced in cows by A7+alum+206 oil, and significantly higher than that by A7+206 oil or inactived vaccines. After FMDV’s attack,4/5 of A7+alum+206 oil immunized cows were protected, whereas half of the cow immunized with inactivated virus vaccine only were protected.Together, based on CpG motif, we designed and screened 63 CpG ODNs containing different motifs and analyze their effect through immune cell proliferation assay. Three AACGTT motif-containing CpG ODNs, designated as YW07, YW08 and YW09, respectively, stood out with vigorous capability of activating canine immune cells. IARV plus YW07 induced more vigorous and lasting specific antibodies in mice and dogs, and protected all of the tested mice from lethal dose of rabies virus challenge. Meantime, we identified RW12 as bovine favored CpG ODN. RW12 could assiste oil-based FMDV recombinant protein A7 to produce high level and lasting antibody in mice or cattle, and protected 4/5 cattle from FMDV challenge. The data suggests that CpG ODN could be used as a novel adjuvant for developing more efficient vaccines of animal use.更多还原