【摘要】：疫苗是經(jīng)濟(jì)有效的預(yù)防傳染病的手段,開發(fā)安全高效的新型疫苗是疫苗學(xué)研究的目標(biāo)。亞單位疫苗和重組蛋白類疫苗的抗原是高純度的蛋白質(zhì),疫苗的安全性得到了提高,但是存在免疫原性弱,激發(fā)的抗體反應(yīng)不強(qiáng),難以激發(fā)T細(xì)胞反應(yīng)等不足。為了提高蛋白類疫苗的效力,研究者們一直在尋找高效的佐劑來增強(qiáng)抗原的免疫原性,誘導(dǎo)更強(qiáng)的免疫反應(yīng)或改變免疫反應(yīng)的類型。鋁佐劑是使用最廣泛、時(shí)間最長的人用疫苗佐劑。近年來,MF59、AS03、AS04和Virosomes等幾種新佐劑逐漸獲批人用。而植物多糖,因其較強(qiáng)的免疫調(diào)節(jié)功能,以及具有生物可降解和低毒性等優(yōu)點(diǎn),成為新型佐劑研究的一個(gè)熱點(diǎn)。茯苓多糖PCP-I是從中藥茯苓中提取純化得到的一種均一多糖,由巖藻糖、甘露糖、葡萄糖和半乳糖組成。本研究以炭疽芽孢桿菌保護(hù)性抗原(PA)為模式抗原,從體液免疫反應(yīng)和細(xì)胞免疫反應(yīng)兩方面對PCP-I的佐劑效應(yīng)進(jìn)行了系統(tǒng)評價(jià);觀察了PCP-I與寡聚脫氧核苷酸CpG構(gòu)成復(fù)合佐劑的佐劑效應(yīng);分別使用炭疽致死毒素(LT)攻毒模型和芽孢攻毒模型,對PCP-I增強(qiáng)PA保護(hù)效力的能力進(jìn)行了考察;從樹突狀細(xì)胞(DC)成熟、生發(fā)中心(GC)反應(yīng)和外周血單核細(xì)胞(PBMC)基因表達(dá)差異三個(gè)角度,對PCP-I佐劑效應(yīng)的機(jī)制進(jìn)行了初步探討。首先對小鼠免疫實(shí)驗(yàn)中PCP-I的量效關(guān)系進(jìn)行了考察。將0.5μg PA分別與50、200、500μg PCP-I混合,免疫BALB/c小鼠,免疫二次,間隔兩周,在二免后兩周取血檢測anti-PA抗體和抗炭疽毒素中和抗體。結(jié)果表明,PCP-I為200μg時(shí),小鼠血清中的antiPA抗體(5.38×103)和中和抗體(8.7×101)顯著高于50μg組(1.54×102,2.65×101),略高于500μg組(4.15×103,6.13×101)。據(jù)此,選擇200μg PCP-I進(jìn)行后續(xù)的實(shí)驗(yàn)。為評價(jià)PCP-I對體液免疫的影響,以0.5μg PA分別混合PCP-I和鋁佐劑(Al),免疫BALB/c小鼠,并設(shè)PA無佐劑組和PBS組分別作為陰性和空白對照,免疫三次,間隔兩周,在三免后兩周取血進(jìn)行相關(guān)檢測。PA+PCP-I組的anti-PA抗體滴度(1.81×104)和抗體親和力(0.81)顯著高于PA組(1.60×103,0.52),而與PA+Al組(2.35×104,0.83)相近。對anti-PA抗體亞類進(jìn)行分析,發(fā)現(xiàn)PA+PCP-I組的IgG1顯著高于IgG2a(5.12×104,2.93×1 03),提示PCP-I是Th2偏向型佐劑。毒素中和實(shí)驗(yàn)結(jié)果表明,PA+PCP-I組的中和抗體(2.55×103)顯著高于PA組(1.32×102),與PA+Al組(1.76×103)無顯著性差異。為評價(jià)PCP-I對細(xì)胞免疫的影響,以5μg PA分別混合PCP-I和鋁佐劑免疫小鼠,PA無佐劑組和PBS組作對照,在三免后兩周取脾細(xì)胞進(jìn)行細(xì)胞免疫相關(guān)檢測。PA+PCP-I組小鼠脾臟中PA特異性記憶B細(xì)胞的頻率(4.26%)顯著高于PA組(0.87%),與PA+Al組(5.39%)無顯著性差異,表明PCP-I能夠增強(qiáng)B細(xì)胞記憶。使用CCK8檢測脾細(xì)胞在體外接受PA刺激后的增殖情況,PA+PCP-I組小鼠脾細(xì)胞增殖指數(shù)(1.37)顯著高于PA+Al組(1.05)和PA組(1.08),表明PCP-I能提高小鼠脾細(xì)胞再次接觸抗原后的增殖能力。使用ELISPOT檢測脾細(xì)胞中IL-4、IFN-γ分泌細(xì)胞的頻率,PA+PCP-I組IL-4分泌細(xì)胞頻率(92/106)高于PA+Al組(34/106),而IFN-γ分泌細(xì)胞的頻率無明顯差異。用細(xì)胞因子芯片對PA刺激培養(yǎng)的脾細(xì)胞上清中各細(xì)胞因子的含量進(jìn)行檢測,PA+PCP-I組IL-2、IL-4、IL-5的含量(63.5、23.0、1020.8pg/ml)顯著高于PA+Al組(17.0、2.6、5.1pg/ml),表明PCP-I增強(qiáng)了記憶T細(xì)胞再次接觸抗原后分泌相應(yīng)細(xì)胞因子的能力。使用炭疽致死毒素(LT)對免疫后小鼠進(jìn)行攻毒,觀察存活率。PA組和PBS組小鼠全部死亡,PA+PCPI組小鼠存活率為75%。為評價(jià)PCP-I與CpG構(gòu)成的復(fù)合佐劑的佐劑效應(yīng),使用0.5μg PA混合不同的佐劑免疫BALB/c小鼠,在三免后兩周取血進(jìn)行檢測。PA+PCP-I+Cp G組的anti-PA抗體(1.02×105)顯著高于PA+PCP-I組(1.81×104)和PA+CpG組(3.49×103);抗體親和力(0.73)顯著高于PA+CpG組(0.52),略低于PA+PCP-I組(0.81)�？贵w亞類分析表明,PA+PCP-I+CpG組的IgG1與PA+PCP-I組相當(dāng)(7.90×104,5.12×104),而IgG2a顯著高于后者(5.12×104,2.93×103),說明聯(lián)合佐劑使得PA特異性體液免疫反應(yīng)呈現(xiàn)Th1/Th2均衡。PA+PCP-I+CpG組的中和抗體(1.38×104)顯著高于PA+PCP-I組(2.55×103)和PA+CpG組(1.62×103)。上述結(jié)果說明復(fù)合佐劑能夠以Th1/Th2均衡的方式提高anti-PA抗體,血清中和毒素的能力也顯著提高。使用LT對小鼠進(jìn)行攻毒,觀察存活率。PA+PCP-I+CpG組的存活率與PA+PCP-I組相同(75%),略高于PA+CpG組(50%)。為更直接地對佐劑增強(qiáng)PA免疫保護(hù)的能力進(jìn)行評價(jià),用0.5μg PA混合不同的佐劑免疫對炭疽芽孢攻擊敏感的A/J小鼠,三免后兩周取血檢測,三免后四周使用炭疽桿菌A16R株芽孢進(jìn)行攻毒。檢測結(jié)果顯示,PA+PCP-I組的anti-PA抗體和中和抗體(7.18×103,2.42×102)低于PA+Al組(3.23×104,3.49×103),而PA+PCP-I+CpG組的抗體和中和抗體(3.23×104,2.29×103)與PA+Al組相當(dāng)。芽孢攻毒后,PA+PCP-I+CpG組和PA+Al組小鼠全部存活,PA+PCP-I組小鼠存活率為83%。PCP-I單獨(dú)使用的佐劑效應(yīng)在A/J小鼠實(shí)驗(yàn)中不如BALB/c小鼠,由于A/J小鼠屬于補(bǔ)體成分5(C5)缺陷型小鼠,推測PCP-I的佐劑效應(yīng)可能與激活補(bǔ)體途徑相關(guān),而CpG的加入能夠在一定程度上彌補(bǔ)C5缺失對PCP-I佐劑效應(yīng)的不利影響。為探討PCP-I的作用機(jī)制,從DC成熟、GC反應(yīng)和PBMC基因表達(dá)差異三個(gè)角度進(jìn)行了初步研究。分別使用PA+PCP-I、PA+Al、PA刺激培養(yǎng)骨髓來源的DC,之后用流式細(xì)胞儀檢測DC表面CD80和MHC-II的表達(dá)。PA+PCP-I刺激組DC中CD80陽性細(xì)胞的比例(82.2%)顯著高于PA組(51.7%),略高于PA+Al組(65.1%);PA+PCP-I組DC中MHC-II陽性細(xì)胞的比例(74.9%)顯著高于PA組(46.8%)和PA+Al組(56.1%)。說明PCP-I能夠誘導(dǎo)DC上調(diào)CD80和MHC-II表達(dá),促進(jìn)DC成熟。使用5μg PA分別混合PCP-I和鋁佐劑免疫BALB/c小鼠,免疫兩次,間隔兩周,在二免后7天取脾細(xì)胞利用流式細(xì)胞術(shù)檢測GC B細(xì)胞和濾泡狀輔助T細(xì)胞(Tfh)的頻率。PA+PCP-I組小鼠脾臟中GC B細(xì)胞(7.73%)頻率顯著高于PA組(6.30%),Tfh細(xì)胞的頻率略高于后者(4.97%vs 4.20%),提示PCP-I能促進(jìn)生發(fā)中心反應(yīng)。使用轉(zhuǎn)錄組測序技術(shù),觀察PA+PCP-I、PA+Al和PA免疫3天后PBMC中基因表達(dá)的差異。通過數(shù)據(jù)分析,PA+PCP-I組和PA組之間比較篩選到66個(gè)差異基因,其中53個(gè)基因與PA+Al組和PA組間差異基因重疊,13個(gè)基因是PCP-I特異的差異基因。對這13個(gè)基因進(jìn)行功能注釋,發(fā)現(xiàn)Il1r2、Clec4e、Stab1和C5ar1與免疫反應(yīng)相關(guān),其中C5ar1編碼C5aR,參與補(bǔ)體C5a對特異性免疫的調(diào)節(jié),與PCP-I的佐劑效應(yīng)與C5相關(guān)的假設(shè)相符。綜上所述,PCP-I作為一種植物多糖類疫苗佐劑,其增強(qiáng)體液免疫反應(yīng)能力與鋁佐劑相當(dāng),增強(qiáng)細(xì)胞免疫反應(yīng)的能力強(qiáng)于鋁佐劑;當(dāng)PCP-I與CpG構(gòu)成復(fù)合佐劑后,以Th1/Th2均衡的方式進(jìn)一步增強(qiáng)了體液免疫反應(yīng);初步的機(jī)制分析發(fā)現(xiàn),PCP-I可通過促進(jìn)DC成熟和增強(qiáng)生發(fā)中心反應(yīng),來增強(qiáng)抗原特異性免疫反應(yīng),其作用機(jī)制還可能與補(bǔ)體C5和C5aR相關(guān)。
[Abstract]:Vaccine is an economical and effective means to prevent infectious diseases. Developing a safe and efficient new vaccine is the target of the study of vaccine. The antigen of subunit vaccine and recombinant protein vaccine is high purity protein. The safety of the vaccine has been improved, but the immunogenicity is weak, the activated antibody reaction is not strong and it is difficult to stimulate the T cell response. In order to improve the effectiveness of the protein vaccine, researchers have been looking for an efficient adjuvant to enhance the immunogenicity of the antigen, to induce a stronger immune response or to change the type of immune response. Aluminum adjuvant is the most widely used and longest human vaccine adjuvant. In recent years, several new adjuvant, such as MF59, AS03, AS04 and Virosomes, have been used. Plant polysaccharide has become a hot spot in the research of new adjuvant because of its strong immune regulation function and its biodegradability and low toxicity. Tuckahoe polysaccharide PCP-I is a kind of homogeneous polysaccharide extracted from Poria cocos, which consists of fucose, mannose, glucose and galactose. This study is based on carbon. Bacillus subtilis protective antigen (PA) is a model antigen, and the adjuvant effect of PCP-I is systematically evaluated from two aspects of humoral immune response and cell immune response. The adjuvant effect of PCP-I and oligo deoxydeoxynucleotide CpG compound adjuvant is observed. The attack model of anthrax lethal toxin (LT) and the spore attack model are used respectively, and PCP-I The ability to enhance the effectiveness of PA protection was investigated. The mechanism of the effect of PCP-I adjuvant effect was preliminarily discussed from three angles: dendritic cells (DC) maturation, germinal center (GC) reaction and peripheral blood mononuclear cell (PBMC) gene expression difference. First, the quantitative effect relationship of PCP-I was investigated in the mice immunization experiment. The 0.5 g PA and 50,2 were respectively compared with 50,2. 00500 micron g PCP-I, immune BALB/c mice, immunized two times, interval two weeks, and two weeks after two immunization to detect anti-PA antibody and anti anthrax neutralization antibody. The results showed that the antiPA antibody (5.38 * 103) and neutralizing antibody (8.7 * 101) in the serum of the mice were significantly higher than that of 50 u g group (1.54 * 102,2.65 x 101), slightly higher than that of the 500 mu g group. 4.15 * 103,6.13 x 101). According to this, 200 mu g PCP-I was selected for subsequent experiments. In order to evaluate the effect of PCP-I on humoral immunity, 0.5 mu g PA was mixed with PCP-I and aluminum adjuvant (Al), and BALB/c mice were immunized with PA without adjuvant and PBS group as negative and blank control, three times of immunization, two weeks interval, and two weeks after three immunity. The titer of anti-PA antibody (1.81 * 104) and antibody affinity (0.81) in.PA+PCP-I group were significantly higher than that in group PA (1.60 x 103,0.52), and similar to that of PA+Al group (2.35 x 104,0.83). The analysis of anti-PA antibody subclass showed that IgG1 in PA+PCP-I group was significantly higher than IgG2a (5.12 x 104,2.93 x 103), suggesting that PCP-I is a biased adjuvant. The results showed that the neutralization antibody (2.55 x 103) in the PA+PCP-I group was significantly higher than that in the PA group (1.32 x 102), and there was no significant difference with the group PA+Al (1.76 x 103). In order to evaluate the effect of PCP-I on cell immunity, the mice were immunized with 5 mu g PA and PCP-I and aluminum adjuvant respectively, and the PA free adjuvant group was compared with the PBS group, and the spleen cells were taken for cellular immune correlation in the two week after three immunity. The frequency of PA specific memory B cells in the spleen of.PA+PCP-I group (4.26%) was significantly higher than that in group PA (0.87%), and there was no significant difference from PA+Al group (5.39%), indicating that PCP-I could enhance the memory of B cells. The proliferation of splenocytes after PA stimulation in vitro was detected with CCK8, and the splenocytes proliferation index (1.37) in PA+PCP-I group was significantly higher than that of PA+Al. Group (1.05) and group PA (1.08) showed that PCP-I could increase the proliferation ability of mouse splenocytes after re contact with antigen. The frequency of IL-4, IFN- gamma secreting cells in splenocytes and the frequency of IL-4 secreting cells in group PA+PCP-I (92/106) were higher than that of group PA+Al (34/106) with ELISPOT, and the frequency of IFN- gamma secretory cells was not significantly different. The content of cytokines in the supernatant of splenocytes in stimulated culture was detected. The content of IL-2, IL-4, and IL-5 in group PA+PCP-I (63.5,23.01020.8pg/ml) was significantly higher than that in group PA+Al (17.0,2.6,5.1pg/ml), indicating that PCP-I enhanced the ability to secrete the corresponding Cytokine after the recontact of the antigen of the memory T cells. The use of anthrax lethal toxin (LT) to immune mice. The survival rate of group.PA and PBS mice was all dead. The survival rate of group PA+PCPI was 75%. as the adjuvant effect of the compound adjuvant of PCP-I and CpG, and the BALB/c mice were immunized with 0.5 mu g PA mixed with different adjuvant. The antibody (1.02 * 105) of.PA+ PCP-I+Cp G group was significantly higher than that of the.PA+ PCP-I+Cp G group (1.02 x 105). Group A+PCP-I (1.81 x 104) and group PA+CpG (3.49 x 103), antibody affinity (0.73) was significantly higher than group PA+CpG (0.52), slightly lower than group PA+PCP-I (0.81). Antibody subclass analysis showed that IgG1 in PA+PCP-I+CpG group was equivalent to group PA+PCP-I (7.90 x 104,5.12 x 104), and IgG2a was significantly higher than that of the latter (5.12 * 104,2.93 x 103), indicating that the combined adjuvant made PA specific body fluids. The immunoreaction showed that the neutralizing antibody (1.38 x 104) of the Th1/Th2 balanced.PA+PCP-I+CpG group was significantly higher than that of the PA+PCP-I group (2.55 x 103) and the PA+CpG group (1.62 x 103). The results indicated that the compound adjuvant could improve the anti-PA antibody in the Th1/Th2 balanced manner and the ability to neutralize the toxin in the serum significantly. The mice were attacked by LT and observed survival. The rate of survival in group.PA+PCP-I+CpG was the same as that in group PA+PCP-I (75%), slightly higher than that in group PA+CpG (50%). It was more direct to evaluate the ability of the adjuvant to enhance the immune protection of PA, with 0.5 mu g PA mixed with different adjuvants to attack the sensitive A/J mice with anthrax spore, three after two weeks of immunization, and three weeks after the use of Bacillus anthracis A16R plant buds. The results showed that the anti-PA and neutralizing antibodies (7.18 x 103,2.42 * 102) of the PA+PCP-I group were lower than that of the PA+Al group (3.23 x 104,3.49 * 103), while the antibody and neutralizing antibody (3.23 * 104,2.29 x 103) in the PA+PCP-I+CpG group were equivalent to that of the PA+Al group. The adjuvant effect of 83%.PCP-I alone is not as good as BALB/c mice in A/J mice. Because A/J mice belong to complement component 5 (C5) deficient mice, it is speculated that the adjuvant effect of PCP-I may be related to the activation of complement pathway, and CpG can make up for the adverse effect of C5 deletion on the PCP-I adjuvant effect to a certain extent. This is to discuss PCP-I. The mechanism of action was preliminarily studied from three angles: DC maturation, GC reaction and PBMC gene expression difference. PA+PCP-I, PA+Al, PA were used to stimulate the culture of DC in bone marrow, and then the expression of CD80 and MHC-II in DC surface was detected by flow cytometry (82.2%) in.PA+PCP-I stimulus group (51.7%), slightly higher than that in the group (51.7%). Higher than group PA+Al (65.1%), the proportion of MHC-II positive cells in group DC (74.9%) in group DC was significantly higher than that in group PA (46.8%) and PA+Al group (56.1%). It indicated that PCP-I could induce DC to increase CD80 and MHC-II expression and promote DC maturation. 5 micron g and aluminum adjuvant immunized respectively, two times, interval two weeks, and splenocytes for 7 days after two immunization. The frequency of flow cytometry was used to detect the frequency of GC B cells and follicular assisted T cells (Tfh) in.PA+PCP-I group. The frequency of GC B cells (7.73%) in the spleen of the mice was significantly higher than that of the PA group (6.30%). The frequency of Tfh cells was slightly higher than that of the latter (4.97%vs 4.20%), suggesting that PCP-I could promote the reaction of the germinal center. The difference of gene expression in post PBMC was analyzed. Through data analysis, 66 different genes were screened from group PA+PCP-I and PA, of which 53 genes overlapped with PA+Al and PA groups, and 13 genes were PCP-I specific differentially genes. The 13 genes were annotated by function, and Il1r2, Clec4e, Stab1 and C5ar1 were associated with immune response, C5ar1, which encodes C5aR, participates in the regulation of complement C5a to specific immunity, which is consistent with the C5 related hypothesis of the adjuvant effect of PCP-I. As a plant polysaccharide vaccine adjuvant, PCP-I has the same ability to enhance the humoral immune response as aluminum adjuvant, which is stronger than the aluminum adjuvant; when PCP-I and CpG constitute a compound. After the adjuvant, the humoral immune response was further enhanced by Th1/Th2 equilibrium. Preliminary mechanism analysis showed that PCP-I could enhance the antigen specific immune response by promoting the maturation of DC and enhancing the reaction of the germinal center, and its mechanism may also be related to the complement of C5 and C5aR.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R392

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