二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

目的 探讨二甲基三十六烷基铵(dimo-thylidioctyl ammonium bromide,DDA)和卡介苗多糖核酸(BCC-PSN)佐剂在结核融合蛋白疫苗加强卡介苗(BCG)免疫中的不同免疫辅助效应.方法 选择DDA、BCG-PSN作为融合蛋白AMM(Ag85B-MPT64190-198-Mtb8.4)的佐剂,在BCG初免小鼠后,AMM疫苗加强免疫两次,其中一组联合使用两种佐剂(DDA/BCG-PSN),另一组单独以DDA作为佐剂,同时设立BCG或磷酸缓冲液(PBS)免疫组为对照.应用ELISA及ELISPOT检测免疫小鼠的体液与细胞免疫反应,最后一次加强免疫后第12周,以H37Rv尾静脉攻毒并检测小鼠肺脾组织细菌载量和病理改变,评价不同佐剂疫苗的保护效果.结果 在BCG初免基础上,联合佐剂组(AMM/DDA/BCG-PSN)和单独佐剂组(AMM/DDA)加强免疫两次后,脾脏淋巴细胞经抗原Ag85B和PPD(purified protein derivative)刺激后,皆可产生分泌较BCG组高的IFN-γ.毒力株攻击后菌落形成单位(colony-forming unit,CFU)计数显示,联合佐剂组(AMM/DDA/BCG-PSN)脾脏荷菌量少于PBS组和BCG组(P<0.05);而单独佐剂组(AMM/DDA)肺部荷菌量少于PBS组和BCG组(P<0.05).组织病理分析结果 表明AMM/DDA/BCG-PSN组肺组织病理损伤较轻,而AMM/DDA组病理损伤个体差异较大.结论 DDA是较为理想的结核亚单位疫苗佐剂,能诱导较强的细胞免疫和免疫保护作用;BCG-PSN可能具有免疫调节作用,可以减轻免疫病理损伤.     

二甲基三十六烷基铵及卡介苗多糖核酸佐剂在结核亚单位疫苗加强BCG免疫中的效应研究

Objective To investigate the adjuvant effect of dimo-thylidioctyl ammonium bromide (DDA) and/or DDA-BCG polysaccharide nucleic acid( BCG-PSN), which was combined with a Mycobacterium tuberculosis fusion protein AMM ( Ag 8 5 B - MPT64190-198 - Mtb8.4 ) to boost BCG primed immunization. Methods DDA with or without BCG PSN was mixed with the fusion protein AMM to construct the boosting vaccine. Mice were immunized with BCG and then boosted twice with AMM formulated with the adjuvant DDA with or without BCG-PSN. PBS or BCG vaccination without boosting was used as control. The humoral and cell-mediated immune responses were analyzed by ELISA and ELISPOT. Moreover, the protective efficacy of BCG prime-AMM subunit vaccine boosting against Mycobacterium tuberculosis infection was analyzed. Results With in vitro stimulation of Ag85B and PPD( purified protein derivative) antigen, the number of IFN-γ secreting cells from the mice boosted twice by AMM/DDA/BCG-PSN and AMM/DDA were higher than BCG and PBS group (P <0.05). The CFU in lungs of mice boosted with AMM/DDA/BCG-PSN was less than that of PBS group(P <0.05), while the CFU of AMM/DDA-boosted mice was less than that of BCG and PBS group(P < 0.05).However, fewer lesions were seen in lungs of mice immunized with BCG alone or BCG-prime-AMM/DDA/BCG-PSN boosting than the other groups. Conclusion DDA is an idea adjuvant for tuberculosis subunit vaccine;BCG-PSN might play a role in alleviating the immunity-mediated pathology.     

CpG-ODN及PolyICLC在结核亚单位疫苗中的佐剂效应

目的观察TLR识别配体不同组合佐剂对结核分枝杆菌融合蛋白免疫原性的影响及DDA对TLR识别配体的辅助效应。方法 CpG-ODN(CpG)和/或PolyICLC联合/不联合DDA,分别与融合蛋白Mtb10.4-HspX(MH)混合,制备亚单位疫苗,于第1、4、7周皮下免疫C57BL/6小鼠。以PBS和BCG(仅免疫1次)作为对照。末次免疫后6周,采血检测血清抗体水平,并分离脾淋巴细胞,检测分泌IFN-γ的淋巴细胞水平。结果经MH及HspX抗原刺激后,MH+CpG+PolyICLC+DDA组小鼠分泌IFN-γ的脾淋巴细胞数高于其它各组(P<0.05),MH+CpG+PolyICLC组其次,显著高于MH+CpG及MH+PolyICLC组(P<0.05);联合DDA佐剂组均分别高于对应的未联合DDA佐剂组(P<0.05)。各亚单位疫苗组诱导产生的抗MH及HspX的IgG1、IgG2b、IgG2c水平均明显高于BCG组(P<0.05),其中MH+CpG+PolyICLC+DDA组3种抗体水平最高;各亚单位疫苗组IgG2c/IgG1均高于BCG组(P<0.05)。结论 CpG+PolyICLC+DDA佐剂增强了结核分枝杆菌融合蛋白的免疫原性;CpG和PolyICLC具有佐剂协同作用;DDA有助于CpG和/或PolyICLC诱导特异性细胞免疫应答。     

结核病蛋白亚单位疫苗佐剂研究进展题录

结核病是由结核分枝杆菌感染引起的全球传染病, 是导致人类死亡的十大主要原因之一。卡介苗(BCG)是目前唯一临床应用的结核病疫苗, 但其对成人结核病的保护作用不明确。在结核病新型疫苗中, 蛋白亚单位疫苗因其成分明确、安全性高的特点, 且具有强化BCG效应的优势, 近年来备受关注。然而, 亚单位结核病疫苗中的蛋白抗原所诱导的免疫应答较弱, 通常需要佐剂辅助以增强其免疫效果。本综述旨在关注结核病疫苗新制剂中使用的各类佐剂(包括已进入临床试验阶段和尚处于临床前开发阶段的佐剂), 对各类佐剂的优缺点及可应用性进行客观评价, 并指出目前佐剂研究中存在的问题及未来的研究方向。     

结核融合蛋白AMM亚单位疫苗强化BCG初始免疫的免疫效应

目的 研究结核融合蛋白Ag85B-Mpt64190-198-Mth8.4(AMM)和佐剂二甲基三十六烷基铵(DDA)、卡介苗多糖核酸(BCG-PSN)构建的亚单位疫苗强化BCG初始免疫的免疫效应.方法 将融合蛋白AMM、佐剂DDA和BCG-PSN混合构建AMM亚单位疫苗.实验1组BCG初免后第10周用AMM亚单位疫苗加强免疫小鼠一次;实验2组BCG初免后分别于第8周、第10周用AMM亚单位疫苗加强免疫小鼠一次.同时设立生理盐水及仅BCG免疫两个对照组.BCG初免后第14周、第22周,应用ELISPOT、ELISA检测免疫小鼠的细胞及体液免疫反应.同时在第22周用BCG活菌攻击被免疫小鼠,间隔4周后用流式细胞术和ELISA技术检测T细胞分型及体液免疫反应.结果 (1)IFN-γ水平:BCG初免后14周,特异性抗原Ag85B刺激后实验2组分泌IFN-γ的细胞数(135±14)明显高于仅BCG免疫组(194±16),t=10.98,P<0.01;BCG初免后22周,实验2组(208±11)同样高于仅BCG免疫组(57±18),t=6.43,P<0.01.(2)体液免疫应答水平:实验2组的IgGI抗体滴度明显高于实验1组,而作为反映Th1型免疫反应指标的IgG2a/IgG1比值,强化免疫两次组低于强化一次组.(3)BCG模拟攻击被免疫小鼠后,CD4+CD25+调节性T细胞含量:实验1、2组均高于仅BCG免疫组(t1=3.08,t<2>=3.16,P<0.05).结论 BEG免疫-AMM亚单位疫苗加强免疫两次能够引起较强的细胞及体液免疫反应,同时激活调节性免疫反应.     

TDM/TDB是结核亚单位疫苗的重要佐剂

结核病是严重危害人类健康的慢性传染病,每年造成全球二百万人死亡[1],我国结核病患者人数仅次印度,位于世界第二位。我国结核病的耐药状况不容乐观,耐多药结核分枝杆菌的出现给结核病的防治带来巨大的挑战[2]。在多数高负担的发展     

亚单位疫苗佐剂的研究进展

亚单位疫苗是通过物理化学方法或基因工程手段获取病原体主要免疫原蛋白而制成的一种疫苗,它可以有效避免利用全病原体生产疫苗所导致的生物安全风险,并有利于提高疫苗中有效成分的浓度、纯度,以及开发多价多联疫苗。目前,已研制成功的亚单位疫苗包括口蹄疫病毒VPI亚单位疫苗、乙肝病毒表面抗原亚单位疫苗、人乳头瘤亚单位疫苗等。     

空肠弯曲菌外膜蛋白亚单位脂质体佐剂疫苗的免疫原性研究

目的 初步探讨自制的空肠弯曲菌（campylobacter jejuni）亚单位（M,28 000-31 000外膜蛋白）脂质体佐剂疫苗的免疫原性,为该疫苗的临床前研究打下基础。方法 用双向免疫琼脂扩散试验、试管凝集试验、ELISA（间接法）分别检测经GJ疫苗免疫后的新西兰兔血清IgG抗体效价。结果 GJ阴离子组与GJ阳离子组比较,血清IgG A值显著增高（P〈0．01）,与GJ弗氏组无统计学差别（P〉0．1）;CJ阴、阳离子组脂质体疫苗接种剂量在0．50mg／kg时,血清IgGA值最大;CJ阳离子皮下注射组与肌肉注射组IgGA值无统计学差别（P〉0．1）;特异性IgG抗体经6个月动态观察,5个月后抗体效价下降。结论 实验组全程免疫后均有特异性抗体产生;同一剂量的阴离子脂质体疫苗比阳离子脂质体疫苗血清抗体水平显著增高;脂质体疫苗最佳接种剂量为0．50mg／kg,肌肉注射与皮下注射免疫效果无显著差异;特异性抗体高水平维持5、6个月后应加强免疫。     

Toll样受体在亚单位疫苗分子设计中的研究进展

近年来固有免疫系统激活与特异性免疫系统活化的关系研究为疫苗分子设计提供了诸多新思路,尤其是人们对Toll样受体了解的不断深化,对疫苗佐剂的发展具有深远影响。Toll样受体除刺激产生针对表位特异性免疫应答外,还具有调节免疫应答类型的能力,从而推动了亚单位疫苗的设计和应用。     

四价流感病毒亚单位疫苗配伍RFH01佐剂在小鼠体内的免疫原性研究

目的评价四价流感病毒亚单位疫苗配伍RFH01佐剂在小鼠模型中的免疫原性。方法按照《中国药典》2020年版(三部)的方法对4种单价流感病毒亚单位疫苗原液进行鉴别试验。在研究配伍RFH01佐剂的四价流感病毒亚单位疫苗小鼠模型中, 采用随机分组法将460只雌性6～8周龄BALB/c小鼠分为实验组、疫苗对照组、阴性对照组和空白组, 共46组, 每组10只。在研究四价流感病毒亚单位疫苗在老年和青年小鼠模型中, 采用随机分组将80只雌性10月龄和80只雌性10周龄BALB/c小鼠同时分为单价流感病毒疫苗、四价流感病毒亚单位疫苗、四价流感病毒亚单位疫苗+RFH01佐剂组、鸡胚四价流感病毒裂解疫苗对照组和PBS组, 共16组, 每组10只。均采用肌内注射免疫, 初次免疫21 d后, 采血分离血清, 并用相同程序和剂量进行加强免疫, 第42天采血分离血清。血凝抑制试验检测小鼠血清抗体水平。结果两次免疫后, 所有配伍RFH01的疫苗组阳转率均达到100%, 且血清抗体效价几何平均滴度(geometric mean titer, GMT)显著高于不含佐剂的疫苗组。配伍RFH01佐剂的单价流感病毒疫苗和四价流...     

DNA vaccine encoding ESAT-6 enhances the protective efficacy of BCG against Mycobacterium tuberculosis infection in mice

ESAT-6 is a protein produced by virulent Mycobacterium tuberculosis but absent in Mycobacterium bovis bacillus Calmette-Guerin (BCG) and is considered as a promising vaccine subunit against tuberculosis. The protective efficacy of a new strategy based on the combination of DNA vaccine encoding ESAT-6 (DNA-E6) and BCG immunization was investigated in the present study. BALB/c mice were immunized with DNA-E6, BCG, DNA-E6 plus BCG, vector plus BCG or vector alone. Three weeks after the last immunization, antigen-specific interferon (IFN)-gamma secreted upon stimulation with BCG-PPD or ESAT-6 protein by splenocytes of the different groups was determined by an ELISA. The immunized mice were challenged intravenously with low-dose virulent M. tuberculosis H37Rv and bacterial load of the infected mice was measured in the spleen and lung 6 weeks later. Pathological changes in the lung were observed by haematoxylin-eosin (HE) or acid-fast staining. Our study demonstrated that ESAT-6-specific IFN-gamma was improved in mice vaccinated with DNA-E6 plus BCG, compared with those mice immunized with BCG alone. The combination of DNA-E6 and BCG resulted in the maximum reduction in bacterial load in both the lung and spleen, and only slight pathological changes were detected in the lung. These results suggested that the combination of DNA-E6 and BCG vaccination could be a better strategy against M. tuberculosis infections in human.     

A novel DNA vaccine for protective immunity against virulent Mycobacterium bovis in mice

Mycobacterium bovis is the causative agent of bovine tuberculosis (bTB). The proteins Ag85B, MPB64, and ESAT-6 are the major immunogenic antigens of M. bovis; these proteins play important roles in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed various DNA vaccines with the genes encoding the three antigens mentioned above. This procedure involved the following steps: fusion of two genes (pcDNA-MPB64-Ag85B, pcMA), fusion of three genes (pcDNA-MPB64-Ag85B-ESAT-6, pcMAE), bivalent combinations (pcDNA-Ag85B + pcDNA-MPB64, pcA + M), and trivalent combinations (pcDNA-Ag85B + pcDNA-MPB64 + pcDNA-ESAT-6, pcA + M + E). The immune response to the DNA vaccines was evaluated based on serum antibody titers, lymphocyte proliferation assay, and titers of the cytokines interferon-γ (IFN-γ) and interleukin-2 (IL-2). The protective efficacy following challenge with a virulent M. bovis strain, C68001, was evaluated based on survival rate, bacterial loads in lung tissue, and histopathologic changes. A significant 2-fold increase in serum antibody levels was observed in mice vaccinated with fusion DNA (two or three genes). Furthermore, the lymphocyte proliferation (SI) values and the levels of IFN-γ and IL-2 were higher in mice vaccinated with fusion DNA (two or three genes) than in those immunized with polyvalent combination DNA vaccines (P < 0.05). Additionally, the fusion DNA vaccines provided protection that was superior to that provided by the polyvalent combination DNA vaccines following challenge with M. bovis strain C68001. The protective efficacy of the fusion DNA vaccines in mice immunized three times was equivalent to the protective efficacy in mice immunized once with the Bacillus Calmette–Guerin (BCG) vaccine. This suggests that fusion DNA vaccine represent a promising approach for the prevention of bTB.     

Exosomes carrying mycobacterial antigens can protect mice against Mycobacterium tuberculosis infection

Approximately 2 billion people are infected with Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB), and an estimated 1.5 million individuals die annually from TB. Presently, Mycobacterium bovis BCG remains the only licensed TB vaccine; however, previous studies suggest its protective efficacy wanes over time and fails in preventing pulmonary TB. Therefore, a safe and effective vaccine is urgently required to replace BCG or boost BCG immunizations. Our previous studies revealed that mycobacterial proteins are released via exosomes from macrophages infected with M. tuberculosis or pulsed with M. tuberculosis culture filtrate proteins (CFP). In the present study, exosomes purified from macrophages treated with M. tuberculosis CFP were found to induce antigen‐specific IFN‐γ and IL‐2‐expressing CD4⁺ and CD8⁺ T cells. In exosome‐vaccinated mice, there was a similar T_H1 immune response but a more limited T_H2 response compared to BCG‐vaccinated mice. Using a low‐dose M. tuberculosis mouse aerosol infection model, exosomes from CFP‐treated macrophages were found to both prime a protective immune response as well as boost prior BCG immunization. The protection was equal to or superior to BCG. In conclusion, our findings suggest that exosomes might serve as a novel cell‐free vaccine against an M. tuberculosis infection.     

Mycobacterium bovis BCG immunization induces protective immunity against nine different Mycobacterium tuberculosis strains in mice

Recent preclinical and epidemiologic studies have suggested that certain Mycobacterium tuberculosis genotypes (in particular, Beijing lineage strains) may be resistant to Mycobacterium bovis BCG vaccine-induced antituberculosis protective immunity. To investigate the strain specificity of BCG-induced protective responses in a murine model of pulmonary tuberculosis, C57BL/6 mice were vaccinated with BCG vaccine and then challenged 2 months later with one of nine M. tuberculosis isolates. Four of these strains were from the W-Beijing lineage (HN878, N4, NHN5, and ChS) while four were non-Beijing-type isolates (C913, CDC1551, NY669, and NY920). As a control, the WHO standard M. tuberculosis Erdman strain was evaluated in these vaccination/challenge experiments. To assess the protective responses evoked by BCG immunization, organ bacterial burdens and lung pathology were assessed in vaccinated and naïve mice at 4, 12, and 20 weeks postchallenge as well as during the day of infection. At 4 weeks after the aerosol challenge with each of these strains, significantly reduced bacterial growth in the lungs and spleens and significantly improved lung pathology were seen in all vaccinated animals compared to naïve controls. After 12 weeks, reduced organ bacterial burdens were detected in vaccinated animals infected with six of nine challenge strains. Although lung CFU values were lower in vaccinated mice for only three of nine groups at 20 weeks postchallenge, significantly decreased lung inflammation was seen in all immunized animals relative to controls at 20 weeks postchallenge. Taken together, these data demonstrate that BCG vaccination protects against infection with diverse M. tuberculosis strains in the mouse model of pulmonary tuberculosis and suggest that strain-specific resistance to BCG-induced protective immunity may be uncommon.     

Enhancing the protective efficacy of Mycobacterium bovis BCG vaccination against tuberculosis by boosting with the Mycobacterium tuberculosis major secretory protein

Tuberculosis continues to ravage humanity, killing 2 million people yearly. Most cases occur in areas of the world to which the disease is endemic, where almost everyone is vaccinated early in life with Mycobacterium bovis BCG, the currently available vaccine against tuberculosis. Thus, while more-potent vaccines are needed to replace BCG, new vaccines are also needed to boost the immune protection of the 4 billion people already vaccinated with BCG. Until now, no booster vaccine has been shown capable of significantly enhancing the level of protective immunity induced by BCG in the stringent guinea pig model of pulmonary tuberculosis, the "gold standard" for testing tuberculosis vaccines. In this paper, we describe a booster vaccine for BCG comprising the purified recombinant Mycobacterium tuberculosis 30-kDa protein, the major secreted protein of this pathogen. In the guinea pig model of pulmonary tuberculosis, boosting BCG-immunized animals once with the 30-kDa protein greatly increased cell-mediated and humoral immune responses to the protein in three consecutive experiments. Most importantly, boosting BCG-immunized animals once with the 30-kDa protein significantly enhanced protective immunity against aerosol challenge with highly virulent M. tuberculosis, as evidenced by a significantly reduced lung and spleen burden of M. tuberculosis compared with those for nonboosted BCG-immunized animals (mean additional reduction in CFU of 0.4 +/- 0.1 log in the lung [P = 0.03] and 0.6 +/- 0.1 log in the spleen [P = 0.002]). This study suggests that administering BCG-immunized people a booster vaccine comprising the 30-kDa protein may enhance their level of immunoprotection against tuberculosis.     

Secretion of functional monocyte chemotactic protein 3 by recombinant Mycobacterium bovis BCG attenuates vaccine virulence and maintains protective efficacy against M. tuberculosis infection

A strain of Mycobacterium bovis BCG that secretes high levels of functional murine monocyte chemotactic protein 3 (BCG(MCP-3)) was developed. Mice vaccinated with BCG(MCP-3) displayed increased lymphocyte migration in vivo and augmented antigen-specific T-cell responses compared to mice vaccinated with BCG alone. The level of protection afforded by BCG(MCP-3) was equivalent to that with control BCG; however, immunodeficient mice infected with BCG(MCP-3) survived significantly longer than mice infected with the control BCG strain. Therefore, BCG(MCP-3) may be a safer alternative than conventional BCG for vaccination of immunocompromised individuals.     

卡介菌多糖核酸注射液效力实验动物模型致敏原的选择

目的针对目前卡介菌多糖核酸注射液效力试验模型不稳定的现状,比较猪血清、牛血清白蛋白、鸡卵清蛋白3种不同致敏原对小鼠的致敏效果,探讨该动物模型致敏原的选择。方法猪血清、冻干牛血清白蛋白、冻干鸡卵清蛋白腹腔注射致敏小鼠,隔天1次,共3次,末次致敏后14d,尾静脉攻击相同的致敏原,观察小鼠的全身发病情况,记录发病指数。结果牛血清白蛋白的致敏效果最差,猪血清和鸡卵清蛋白致敏效果好,但是反复冻融后的猪血清致敏效果较猪血清效果显著性降低（P〈0．05）。鸡卵清蛋白的致敏浓度选择结果,在5mg/ml的攻击浓度下,动物的发病指数0．5mg／ml致敏浓度组高于0．25mg／ml剂量组和1mg／ml剂量组。结论鸡卵清蛋白更适合用于卡介菌多糖核酸注射液效力试验的致敏原,最佳的致敏浓度和攻击浓度分别为0．5mg／ml和5mg／ml。     

Immunogenicity and protective efficacy of a fusion protein vaccine consisting of antigen Ag85B and HspX against Mycobacterium tuberculosis infection in mice

Subunit vaccines have the potential advantage to boost Mycobacterium bovis Bacillus Calmette‐Guérin (BCG)‐primed immunity in adults. However, most candidates are antigens highly expressed in replicating bacilli but not in dormant or persisting bacilli, which exist during Mycobacterium tuberculosis infection. We constructed M. tuberculosis fusion protein Ag85B‐Mpt64_190–198‐HspX (AMH) and Ag85B‐Mpt64_190–198‐Mtb8.4 (AMM), which consist of Ag85B, the 190–198 peptide of Mpt64, HspX (Rv2031c) and Mtb8.4 (Rv1174c), respectively. AMH and/or AMM were mixed with adjuvants composed of dimethyl‐dioctyldecyl ammonium bromide and BCG polysaccharide nucleic acid (DDA‐BCG PSN) to construct subunit vaccines. Mice were immunized thrice with Ag85B, AMH and AMM vaccines and the immunogenicity of the fusion protein vaccines was determined. Then, mice were primed with BCG and boosted twice with Ag85B, AMH, AMM and AMM + AMH vaccines, respectively, followed by challenging with M. tuberculosis virulent strain H37Rv, and the immune responses and protective effects were measured. It was found that mice immunized with AMH vaccine generated high levels of antigen‐specific cell‐mediated responses. Compared with the group injected only with BCG, the mice boosted with AMM, AMH and AMM + AMH produced higher levels of Ag85B‐specific IgG1 and IgG2a and IFN‐γ‐secreting T cells upon Ag85B and Mycobacterium tuberculosis purified protein derivative (PPD) stimulation. It is interesting that only mice boosted with AMM + AMH had significantly lower bacterial count in the lungs than those receiving BCG, whereas mice boosted with AMH or AMM did not. The results suggest that AMH consisting of HspX, the antigen highly expressed in dormant bacilli, could be combined with antigens from replicating bacilli to enhance BCG primed immunity so as to provide better protection against both growing and non‐growing bacteria that occur during the infection process.