国产b型流感嗜血杆菌结合疫苗免疫原性观察

目的:了解兰州生物制品研究所研制的b型流感嗜血杆菌结合疫苗的免疫原性。方法:采用单纯随机抽样方法对柳州市709名3～59月龄婴幼儿按照规定的免疫程序接种兰州所(或巴斯德)Hib疫苗,2年内在不同时段采集血清样本,采用ELISA方法检测Hib-PRP抗体滴度。结果:基础免疫后血清Hib-PRP抗体含量平均水平为18.043μg/ml,抗体阳转率为97.19%,1年后血清Hib-PRP抗体含量为7.575μg/ml,抗体阳转率为93.75%。加强免疫后血清Hib-PRP抗体含量为130.330μg/ml,抗体阳转率为100.00%,加强免疫1年后血清Hib-PRP抗体含量为51.723μg/ml,抗体阳转率为100.00%。基础免疫后接种巴斯德Hib疫苗组血清Hib抗体含量高于接种兰州所Hib疫苗组,抗体阳转率则没有差别;免后1年,接种兰州所Hib疫苗儿童体内Hib抗体含量平均水平高于接种法国巴斯德Hib疫苗儿童;其余各时段两组儿童血清Hib-PRP抗体含量及抗体阳转率均无差别。结论:兰州所Hib疫苗有较好的免疫原性和免疫持久性。     

3批Hib结合疫苗的批间一致性、安全性和免疫原性:婴儿中随机、多中心Ⅲ期临床研究结果

正针对流感嗜血杆菌b型(Hib)的疫苗接种隶属于美国的常规儿科免疫计划。以前由于疫苗的短缺,因此需要另外的Hib疫苗接种方案。此次Ⅲ期随机、多中心研究(NCT01000974)评估了单价Hib-破伤风类毒素结合疫苗(Hib-TT)与单价(Hib-TT对照)、Hib-TT联合疫苗相比较的安全性和免疫原性。作者分层评估了3个Hib-TT批次的批间一致性和Hib-TT对Hib-TT对照的非劣效     

伤寒Vi-rEPA结合疫苗在小鼠中的免疫原性研究

以伤寒Vi多糖疫苗及结合物含量分别为20％、40％、60％的伤寒Vi-rEPA结合疫苗经皮下免疫小鼠后眼眶采血分离血清,用ELISA法测血清抗体浓度。血清抗体分析表明,伤寒Vi多糖疫苗和伤寒Vi-rEPA结合疫苗均有良好的免疫原性,但伤寒Vi-rEPA结合疫苗的免疫原性优于伤寒Vi多糖疫苗;伤寒Vi多糖疫苗无加强免疫应答效应,而伤寒Vi-rEPA结合疫苗有加强免疫应答效应;不同高分子结合物含量的伤寒Vi-rEPA结合疫苗免疫小鼠,其结合物含量为40％、60％的伤寒Vi-rEPA结合疫苗比结合物含量为20％的结合疫苗具有更好的免疫原性,而前二者的免疫原性无显著性差异。     

鼠伤寒结合疫苗研制的初步探讨

通过对鼠伤寒沙门菌LH株的发酵培养,热酚水法提取脂多糖LPS,1%乙酸沸水浴水解90m in脱毒,Super-dex 200柱层析,收集第一峰为鼠伤寒O-SP抗原;然后用CDAP对O-SP活化、ADH衍生后,在EDAC的缩合作用下,结合到破伤风类毒素TT上,制备出鼠伤寒结合疫苗;用含2.5μg多糖鼠伤寒结合疫苗免疫小鼠,以2.5μgO-SP多糖生理盐水溶液以及生理盐水溶液为对照组,间隔14天,免疫三针;以LPS为包被抗原,用间接ELISA法测定血清中抗鼠伤寒LPS IgG抗体。鼠伤寒结合疫苗三针免疫后,小鼠血清抗鼠伤寒LPS IgG抗体效价达到1:80以上的比例为84.2%,而总的几何平均滴度(GMT)达到796;说明制备的鼠伤寒结合疫苗有良好的免疫原性,而且鼠伤寒结合疫苗在小鼠和豚鼠体内有良好的安全性。     

不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平分析

目的研究伤寒Vi多糖蛋白结合疫苗免疫效果及不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体水平,以确定合适的免疫剂量。方法将150只清洁级NIH雌性小鼠随机分为5组,分别为A组(0.625μg结合疫苗组)、B组(1.250μg结合疫苗组)、C组(2.500μg结合疫苗组)、D组(2.500μg多糖组)及阴性组(10mmol/L PBS),每组30只;另领取10只为空白对照(不接种)。A、B、C、D组及阴性组小鼠经腹股沟皮下注射,剂量0.1 m L/只,每隔2周免疫1次,共免疫3次,每次免疫后第7天采血。采用ELISA检测小鼠血清抗体效价,同时对不同剂量伤寒Vi多糖蛋白结合疫苗在小鼠体内诱导的抗体效价进行分析比较。结果与D组相比,A、B、C三组诱导的抗体水平与之均有统计学意义(P<0.05);A组与B组、A组与C组之间的抗体水平也具有统计学意义(P<0.05),而B组与C组之间的抗体水平无统计学意义(P>0.05)。说明与多糖疫苗相比,伤寒Vi多糖蛋白结合疫苗能够诱导更高的抗体水平,且具有明显的剂次加强效应。同时证明1.250μg的伤寒Vi多糖蛋白结合疫苗可诱导与2.500μg伤寒Vi多糖蛋白结合疫苗相同的抗体水平。结论伤寒Vi多糖蛋白结合疫苗的两种免疫剂量在小鼠体内可诱导相同的抗体水平,在选择接种剂量时,可致免疫应答的无统计学意义的低剂量可能是较为经济和安全的选择。     

玉竹多糖对流感病毒裂解疫苗的黏膜佐剂效应

探讨玉竹多糖(Polygonatum odoratun polysaccharides,POP)对流感病毒裂解疫苗黏膜免疫的佐剂效果。以BALB/c小鼠为模型,将H7N9流感病毒裂解疫苗单独或者添加不同剂量的POP(400、600、800、1 000和1200μg)做佐剂一次性滴鼻免疫小鼠,免疫三周后小鼠以致死剂量同源病毒攻击。实验结果表明,当在疫苗中加入1 000μg POP时,与疫苗单独免疫相比,单次滴鼻免疫就明显增强了小鼠抵抗致死量病毒感染的能力,体重丢失减少,肺部残余病毒滴度下降,存活率提高;血清中疫苗特异性IgG抗体和HI抗体以及鼻洗液中SIgA抗体都提高了;IgG亚类抗体检测显示,POP的加入显著增加了IgG1的抗体滴度,但抑制了IgG2a的产生,说明诱导的免疫应答偏向于Th2型。实验表明POP在一定剂量时能增强疫苗诱导的免疫应答,显示出黏膜佐剂效应。     

EV71与CA16双价灭活疫苗诱导小鼠免疫原性研究

目的评价EV71和CA16双价灭活疫苗免疫小鼠诱导的体液免疫和细胞免疫应答。方法分别应用EV71和CA16双价疫苗、EV71和CA16单价疫苗按单针、两针(0,14 d)的程序免疫小鼠,采用细胞病变法检测血清第1针免疫后21 d时中和抗体效价,应用LUMINEX液相芯片技术检测小鼠脾单个核细胞体外经抗原刺激分泌细胞因子的水平。结果双价疫苗与EV71单价疫苗单针、两针免疫相比,EV71中和抗体几何平均值相近(118.8∶84.0;159.5∶156.8,P0.05)。双价疫苗与CA16单价疫苗单针免疫诱导的CA16中和抗体均为阴性;两针免疫CA16中和抗体几何平均值一致(30.0∶26.9,P0.05)。加强免疫7 d后,小鼠脾MNC经EV71抗原刺激,双价疫苗较EV71单价疫苗诱导Th2类细胞因子IL-4、5、6、10和炎症因子TNF-α的分泌水平显著增高(P=0.020,P=0.027,P=0.038,P=0.019,P=0.026);MNC经CA16抗原刺激,双价疫苗与CA16单价疫苗相比诱导细胞因子水平差异无统计学意义(P0.05)。结论双价疫苗可诱导与单价疫苗相似的中和抗体应答水平,并可提高Th2类细胞因子应答,研究为EV71和CA16双价灭活疫苗的研发提供了实验依据。     

C群脑膜炎球菌结合物小鼠免疫原性的研究

目的比较C群脑膜炎球菌多糖蛋白质结合物(简称结合物)的相对分子质量大小和免疫剂量对其在小鼠体内免疫原性的影响,为结合物的分子大小的质控和结合疫苗成品免疫剂量的选择提供实验依据。方法利用CDAP活化法制备C群脑膜炎球菌结合物,通过硫酸铵盐析进行纯化,然后利用Sepharose CL-4B凝胶过滤层析分析,并根据化学检测结果将结合物分为KD0-0.2(组分1)、KD0.2-0.4(组分2)、KD0.4-0.7(组分3)等3个不同相对分子质量组分,每份分别采用1.0μg、0.2μg的免疫剂量免疫小鼠,并对血清进行ELISA分析。结果采用1.0μg免疫剂量时,3种不同相对分子质量的C群脑膜炎球菌结合物均能产生较高水平的抗体,具有典型的加强效应,第2剂免疫即可产生高水平的抗体,而第2、3剂免疫后的抗体水平差异无统计学意义;采用0.2μg免疫剂量时,三种不同相对分子质量的C群脑膜炎球菌结合物只有在第3剂免疫后才能产生较高水平抗体,第1、2剂免疫后的抗体水平差异无统计学意义,组分1和组分2在第3剂免疫后产生的抗体水平优于组分3;对于相同相对分子质量结合物,用1.0μg免疫小鼠产生的抗体水平优于0.2μg免疫组,而且有显著的统计学意义。结论高剂量时,多糖相对分子质量大小对C群脑膜炎球菌结合物的免疫原性没有显著性影响;低剂量时,小相对分子质量结合物对其免疫原性有影响。同等相对分子质量时,1.0μg比0.2μg的免疫剂量可以激发更高的抗体水平。     

中国HIV-1 B亚型P55和嵌合的P55-V3病毒样颗粒候选疫苗免疫效果的研究

研究我国艾滋病病毒Ⅰ型（ＨＩＶ－１）病毒样颗粒（ＶＬＰ）候选疫苗的免疫原性,为疫苗进行灵长类实验提供实验依据。将ｇａｇ和ｇａｇ－Ｖ３ ＶＬＰ不同剂量（５０μｇ、１０μｇ、１μｇ）在有佐剂（氢氧化铝）和无佐剂条件下皮下免疫小鼠,然后眼眶采血,用ＥＬＩＳＡ法观察免疫鼠血清中抗体与剂量关系,以及中和抗体滴度和抗体ＩｇＧ亚型ＩｇＧ１和ＩｇＧ２ａ的水平。同时取鼠脾淋巴细胞,体外抗原刺激后收取淋巴细胞分泌上清     

免疫刺激复合物疫苗制备及其对小鼠免疫功能的影响

确定了免疫刺激复合物(ISCOM)疫苗的安全有效剂量及其对小鼠免疫功能的影响。选取体重25 g左右的昆明小白鼠60只,分为12组,每组5只,腹腔分别注射不同剂量(5-200μg)的ISCOM疫苗,观察小鼠健康状态。选取体重28 g左右的昆明小白鼠60只,分为4组,分别在腹腔注射相同剂量的灭菌生理盐水,Lipase+生理盐水,空ISCOM,Lipase+ISCOM疫苗,利用间接ELISA检测血清中特异性抗体效价。结果表明,当注射剂量为5-25μg/只时,小鼠无任何异常症状。免疫后8 d,小鼠血清特异性抗体效价(log2)可以达到10.5,显著高于对照组。因此,ISCOM疫苗能有效引起小鼠的免疫反应。     

ELISA方法测定血清中Hib多糖抗体含量的条件比较研究

分别以牛血清白蛋白和人血清白蛋白作为封闭液的主要成分测定7份血清中抗b型流感嗜血杆菌荚膜多糖的抗体含量,结果显示,两组数据之间没有显著性差异(P>0.05)。分别用HRP标记的羊抗人IgG和HRP标记的鼠抗人IgG测定该7份血清,结果显示两组结果无显著性差异(P>0.05)。同时,将血清反应的温度和时间由37℃1.5h改为4℃16h(过夜),结果显示两者无显著性差异(P>0.05)。从而优化了该方法。     

人参多糖对新甲型H1N1流感病毒灭活疫苗的免疫增强作用

在流感灭活疫苗中添加佐剂可以提高疫苗的免疫原性,节约抗原用量。一些天然中草药多糖具有潜在的佐剂效应。本文探讨了人参多糖（ginseng polysaccharide,GPS）在新甲型H1N1流感病毒裂解型灭活疫苗中的佐剂效应。将不同剂量GPS与新甲型H1N1流感病毒灭活疫苗混合,共同免疫小鼠一次,通过检测免疫后在小鼠体内诱导产生的疫苗特异性IgM、IgG、IgG1和IgG2a抗体情况来评价GPS作为流感病毒灭活疫苗佐剂的免疫增强效果,并与不添加佐剂的疫苗和加有铝佐剂的疫苗的免疫效果作比较。结果显示,GPS与铝佐剂一样能显著提高和维持疫苗特异性IgG抗体滴度,同时提高IgM抗体水平,其中800μgGPS的佐剂效果最好。因此我们认为GPS可以作为流感病毒灭活疫苗的一种候选佐剂。     

Process development and immunogenicity studies on a serogroup ‘X’ Meningococcal polysaccharide conjugate vaccine

Meningococcal group X (MenX) is responsible for recent outbreaks of meningitis reported in sub-Saharan region of Africa. Although protective vaccines are available for meningitis, they are not effective against MenX. An efficacious, monovalent conjugate vaccine was designed against MenX and a fed-batch fermentation process was developed. The MenX polysaccharide (PS) was purified and yield estimated to be 15-fold higher than the reported elsewhere. Structure of MenX polysaccharide was confirmed by ¹H, ¹³C NMR spectroscopy analysis. Molecular weight of PS was found to be 310 kDa using HPLC-SEC coupled to refractive index (RI) detector. The MenX–Tetanus toxoid (TT) monovalent conjugate proved to be highly immunogenic in mice, and the bactericidal titers of MenX–TT conjugate were 10-fold higher than native PS. Increasing the dose of MenX–TT conjugate from 0.5 μg to 1.0 μg induced an 8-fold higher antibody titer as well as serum bactericidal titer. The current work suggests that the MenX–TT conjugate is a candidate vaccine against meningitis caused by Meningococcal group X strains.     

Mutual interactions between DTaP-IPV and Haemophilus influenzae type b (Hib)-conjugated vaccines in laboratory animal models.

Potency and/or immunogenicity of three different Haemophilus influenzae type b-conjugated vaccines (Hib) and a DTaP-IPV vaccine alone, and their mutual interactions in DTaP-IPV-Hib combination was tested. In a mouse model, only combination of Act-Hib, in which tetanus toxoid (TT) was as active as non-conjugated TT, significantly increased the immunogenicity and potency of TT component of DTaP-IPV vaccine. Also, only combination of Hib-TITER, in which CRM197 was used as the carrier with DTaP-IPV, increased the potency of diphtheria toxoid (DT) component of DTaP-IPV vaccine significantly. It shows that the additive effect of tested Hib vaccines on immunogenicity and/or potency of TT and DT was mostly due to the existence of TT and CRM197, respectively, as the carrier in the mentioned Hib vaccines. No difference was shown in inoculation of DTaP-IPV and Hib conjugated vaccines in the same syringe or at separate sites. DTaP-IPV had dual effects on anti-Hib capsular polysaccharide (HibCP) responses to Hib vaccines in the mouse model. This duality was probably related to the carrier B-cell epitopes activity of Hib conjugated vaccines. The immunogenicity of TT component of Act-Hib and Amvax Hib-TT in the guinea pig model was shown and combination of mentioned Hib vaccines with DTaP-IPV, remarkably increased anti-TT antibody responses to the TT component of DTaP-IPV vaccine. These confirmed our results in the mouse model. Using two different protocols to evaluate the guinea pig model for induction of anti-HibCP immunity showed that a "long interval" protocol does not have any advantage over the "short interval" protocol. Also, combination of DTaP-IPV with Hib vaccines did not have any noticeable effect on anti-HibCP antibodies in the guinea pig model. Taken together, our observations in laboratory animal models may facilitate a better understanding of the mutual interactions between the different antigen components of a combined vaccine such as DTaP-IPV-Hib vaccine.     

Experimental design and metabolic flux analysis tools to optimize industrially relevant Haemophilus influenzae type b growth medium

Haemophilus influenzae type b (Hib), a Gram‐negative capsulated bacterium, is a causative agent of meningitis worldwide. The capsular polysaccharide, a high molecular mass polymer consisting of the repeated units of the polyribosyl‐ribitol‐phosphate, is considered the main virulence factor and it is used as an antigen to vaccines, conjugated to a carrier protein. The industrial production of the polysaccharide requires the cultivation of Hib in rich medium, which impacts process costs and product recovery. In this study, a central composite rotational experimental design strategy was used to access the influence of key components of culture medium (soy peptone, yeast extract and glucose) on biomass formation and polysaccharide production in shake‐flasks. The optimized medium formulation, containing half of the usual yeast extract and soytone concentrations, was further validated in batch bioreactor cultivations. High polysaccharide production (～500 mg/L) was obtained in a cheaper and more competitive production process for use in Hib vaccine production. In addition, simulations of a metabolic model describing Hib central metabolism were used to assess the role of key amino acids on growth. A chemically defined medium supplemented only with amino acids from α‐ketoglutarate and oxaloacetate families as well as phenylalanine was suggested as a promising alternative for reduced acetate accumulation and enhanced polysaccharide production in Hib cultures. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1508–1519, 2017     

b型流感嗜血杆菌结合疫苗冻干剂型的研究

对b型流感嗜血杆菌结合疫苗冻干剂型进行了研制。选用2.5%的甘氨酸作为稳定剂对疫苗进行冻干,对冻干疫苗的抗原性、免疫原性和安全性等理化特性和免疫学特性进行了检定,并与同类疫苗进行比较。各项检定结果及系统观察表明:冻干疫苗安全性良好,抗原性及免疫原性没有发生改变。与同类疫苗相比,针对HibCPS抗原的IgG抗体及针对TT抗原的抗体水平具有相同的变化趋势。冻干疫苗稳定性较冻干前明显变好,效期可延长至3年;不仅有利于保存和运输,而且方便与其他疫苗联合使用。     

Presence of multiple copies of capsulation loci in invasive Haemophilus influenzae type b (Hib) strains in Japan before introduction of the Hib conjugate vaccine

Despite the effectiveness of the Hib vaccine, multiple amplification of the capb locus contributes to vaccine failure. However, there has been no report on the effect of Hib locus amplification in Japan. We examined 24 Hib strains from Japanese children with invasive diseases due to Hib. Although all strains showed the same capb sequence, Southern blot analysis showed that four strains (16.7%) harbored multiple copies (more than two) of the capb locus. Careful analysis of the locus in circulating Hib strains is necessary now that the Hib vaccine has been introduced into Japan.     

rHB-DTaP联合疫苗中抗-HBs抗体应答

在乙肝、白喉、破伤风、无细胞百日咳（ｒＨＢ－ＤＴａＰ）四联疫苗的研制过程中,我们采用基因工程乙肝疫苗（ＣＨＯ）,精制白喉、破伤风类毒素和无细胞百日咳菌苗原液,按不同的稀释液,不同的加入顺序以及不同的含量等制备四联疫苗,并以单价基因工程乙肝疫苗（ｒＨＢ）做对照,进行小鼠试验,接种４周后采血检测抗－ＨＢｓ水平。结果表明,不同配方的四联疫苗中ｒＨＢ与ＤＴａＰ均无干扰,相容性好;抗－ＨＢｓ免疫应答水平与单价苗（ｒＨＢ）无显著性差异（Ｐ＞０．０５）,使用生理盐水制备的ｒＨＢ－ＤＴａＰ其抗－ＨＢｓ免疫应答水平较醋酸盐缓冲液者高（Ｐ＜０．０５）,ｒＨＢ－ＤＴａＰ中的抗－ＨＢｓ免疫应答水平与ｒＨＢ原液的加入顺序无关（Ｐ＞０．０５）;ｒＨＢ含量以２０μｇ／ｍｌ为宜。     

2～59岁健康人群接种ACYW135群脑膜炎球菌多糖疫苗的免疫原性观察

目的评价ACYW135群脑膜炎球菌多糖疫苗在2～59岁健康人群中的免疫原性。方法 2～59岁健康人群接种者随机抽样(n=60),接种一剂四价脑膜炎球菌多糖疫苗。采集接种前和接种后1个月血清,采用体外杀菌试验(Serum bactericidal assay,SBA)检测血清中抗A、C、Y、W135群脑膜炎球菌的血清杀菌滴度。结果免疫前、后血清抗A群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为1241(736,2091)和7559(5520,10351)(P<0.05);抗C群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为4(9,21)和4787(2947,7775)(P<0.05);抗W135群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为16(9,28)和368(162,883)(P<0.05);抗Y群脑膜炎球菌的血清杀菌滴度GMTs(95%CI)分别为120(58,246)和1373(687,2745)(P<0.05)。免疫前和免疫后血清抗A群脑膜炎球菌的杀菌滴度≥128的比例分别为87(77.4,95.1)%和100(83.2,100)%;抗C群脑膜炎球菌的比例分别为17(8.3,28.5)%和97(88.5,99.6)%;抗W135群脑膜炎球菌的比例分别为13(5.9,24.6)%和68(55.0,79.7)%;抗Y群脑膜炎球菌的比例分别为57(43.2,69.4)%和85(73.4,92.9)%。免疫后较免疫前抗A群、C群、W135群和Y群脑膜炎球菌杀菌抗体滴度≥4倍升高的比例分别为50(27.2,72.8)%、97(88.5,99.6)%、62(43.2,73.9)%和55(41.6,67.9)%。结论虽然免疫前人群由于地方和国家免疫计划的实施已具有较高水平的抗A群脑膜炎球菌的血清杀菌滴度,但接种ACYW135群脑膜炎球菌多糖疫苗后可以使其保护水平进一步提高,并使人群对C群、W135群和Y群脑膜炎球菌的低水平杀菌抗体滴度均显著升高达到保护水平,证明ACYW135群脑膜炎球菌多糖疫苗在2～59岁健康人群中具有比较好的免疫原性。     

b型流感嗜血杆菌结合疫苗中载体蛋白质的效力研究

考察b型流感嗜血杆菌(Hib)结合疫苗的载体蛋白质—破伤风类毒素(TT)的免疫原性,为百白破与Hib四联疫苗中TT的使用提供参考。方法:将小鼠随机分为四组,分别注射Hib结合疫苗、Hib与百白混合疫苗、Hib与百白破混合疫苗、破伤风类毒素,比较它们在NIH小鼠体内所诱导产生的特异性抗体水平,并对这四组疫苗进行破伤风效力保护试验。结果表明,破伤风类毒素组,Hib与百白破混合疫苗组刺激的TT抗体水平远大于Hib结合疫苗组和Hib与百白混合疫苗组。效力试验虽四组间没有差异,只说明一定的抗体量就可以对小鼠提供保护。认为Hib结合疫苗中的载体蛋白并不能替代百白破疫苗中的破伤风类毒素。