测定脑膜炎球菌疫苗C群多糖的双抗体夹心ELISA法的建立

目的 建立测定A、C、Y、W135群脑膜炎球菌多糖疫苗(groups A,C,Y,W135 meningococcal polysaccharide vaccine,MPV4)C群多糖含量的双抗体夹心ELISA法.  方法 制备抗C群多糖多克隆抗体,所得的多克隆抗体经辛酸-硫酸铵沉淀法纯化后,用过碘酸钠法对其标记辣根过氧化物酶.分别将抗C群多糖多克隆抗体作为包被抗体和酶标二抗,建立双抗体夹心ELISA法,优化反应条件,对C群多糖进行特异性定量测定.  结果 建立的双抗体夹心ELISA法特异性较好,未检出与A、Y、W135群多糖的交叉反应.C群多糖在2.5～20.0 ng/ml质量浓度范围的剂量反应曲线线性最佳,相关系数大于0.99.该法的准确度和精密度均较好,试验内和试验间变异系数和多糖回收率分别为0.6％ ～9.1％和87.5％ ～ 100.0％,定量限度为4.0 ng/ml.采用该法测定3批MPV4显示,C群多糖含量及多糖分子大小KD值和回收率的测定结果均与先前的测定结果一致,均符合暂行质量标准.  结论 建立的双抗体夹心ELISA法可用于MPV4 C群多糖的关键质量指标测定.     

检测脑膜炎球菌多糖疫苗Y群多糖含量和分子大小的双抗体夹心ELISA法的建立和初步应用

目的建立测定A、C、Y、W135群脑膜炎球菌多糖疫苗（groupsA，C，Y，W135meningococ—calpolysaccharidevaccine，MPV4）Y群多糖含量的双抗体夹心ELISA法。方法采用杂交瘤技术制备抗Y群多糖单克隆抗体，并通过过碘酸钠法用辣根过氧化物酶标记单克隆抗体。分别以抗Y群多糖不同位点的单克隆抗体作为包被抗体和酶标二抗，通过优化反应条件来建立双抗体夹心ELISA法，同时进行方法学验证。结果建立的双抗体夹心ELISA法特异性良好，未检出与A、C、W135群多糖的交叉反应。Y群多糖在2．5～20．0ng／ml范围的剂量反应曲线呈现最佳线性关系，相关系数〉0．99。该法的试验内和试验间准确度较好，精密度较佳，定量限度为4ng／ml。采用该法测定3批MPV4Y群多糖显示，Y群多糖的含量、多糖分子大小‰值和回收率的结果均与先前的检定结果一致，符合暂行质量标准。结论建立的双抗体夹心ELISA法可用于MPV4Y群多糖的关键质量指标测定。     

火箭免疫电泳法定量检测4价脑膜炎球菌多糖疫苗中C群脑膜炎球菌多糖含量的方法学验证

目的 验证用火箭免疫电泳法(rocket immunoelectrophoresis,R1E)检测4价脑膜炎球菌多糖疫苗(tetravalent serogroup A／C／W135／Y meningDcoccal polysaccharide vaccine,MPV4)中C群多糖含量的可靠性。方法 以系列浓度的C群多糖溶液为标准,采用RlE对MPV4中C群多糖含量迸行重复测定。结果 最佳线性范围为30～86 mg／L,相关系数(r)均大于0.985;MPV4与C群多糖参比品的剂量反应曲线之间具有良好的平行性;在精密度试验中,试验内CV为6.08％～8.07％,试验间Cy为7.24％～9.19％;A、W135和Y群多糖及乳糖不引起非特异性免疫反应。结论 本法的线性、精密度和专属性均符合验证要求,可作为定量检测MPV4中C群多糖含量的方法。     

火箭免疫电泳在W135群脑膜炎球菌发酵工艺优化中的应用

目的  应用火箭免疫电泳（rocket immunoelectrophoresis，RIEP）检测W135群脑膜炎球菌（group W135 meningococcus，MenW135）发酵液的多糖含量，来指导MenW135发酵工艺的改进。方法  制备家兔MenW135抗血清，并确定RIEP琼脂糖凝胶中的抗血清浓度。分别以不同发酵条件培养MenW135，并分别在培养2、3、4、5、6 h时取样，用分光光度法检测菌液的吸光度（A），用RIEP检测菌液的多糖含量。结果  检测MenW135多糖的RIEP琼脂糖凝胶中的抗血清浓度确定为0.1%。在MenW135发酵2~5 h期间，MenW135多糖含量与其A值呈正相关性。然而，在MenW135发酵5~6 h期间，菌液的A值不增加，但菌液的多糖含量仍在升高。结论  相对于通过菌液的A值来观察菌液的多糖含量，RIEP检测能直接获得菌液的多糖含量，从而更好地指导MenW135多糖疫苗发酵工艺的优化。     

层析法提纯A、C、Y、W135群脑膜炎球菌荚膜多糖工艺的建立

目的 建立A、C、Y、W135群脑膜炎球菌荚膜多糖层析纯化工艺。方法 采用陶瓷羟基磷灰石和DEAE Sepharose FF层析柱，在PBS体系中纯化A群多糖。以0.5%脱氧胆酸钠预处理C群多糖，用陶瓷羟基磷灰石层析柱在PBS体系中纯化C群多糖。以含有脱氧胆酸钠的平衡缓冲液溶解Y和W135群多糖，再用Capto Adhere和Capto DEAE层析柱串联纯化。纯化的多糖经Sephadex G-25 Medium层析柱脱盐后冻干，按中国药典2015年版三部的要求进行检定。结果  经过层析纯化，A、C、Y、W135群多糖的蛋白质含量分别降至3.7、4.2、5.4和5.3 mg/g，核酸含量分别降至1.2、3.0、1.1和0.8 mg/g，均符合药典要求。此外，磷、唾液酸和O-乙酰基含量等指标亦均符合药典标准。结论 建立了A、C、Y、W135群脑膜炎球菌荚膜多糖的层析纯化工艺。     

ACYW135群脑膜炎球菌多糖疫苗的研制

目的 通过研究冷酚与粗糖的混合比例、粗糖提纯时的质量浓度和乳糖保护剂的用量来制备符合规定的ACYW135群脑膜炎球菌多糖疫苗.方法 发酵细菌,提取A、C、W135、Y群脑膜炎球菌多糖粗制品,以不同的纯化工艺纯化A、C、W135、Y群脑膜炎球菌多糖.将4种纯化的多糖原液按一定比例混合,加入乳糖作为保护剂,冷冻干燥制成ACYW135群脑膜炎球菌多糖疫苗.结果 通过比较不同纯化工艺制备的多糖,确定最佳提纯条件为多糖与冷酚的体积比为1∶1,粗糖提纯时的质量浓度为6 g/L.检测按此纯化条件制备的ACYW135群脑膜炎球菌多糖疫苗显示,3批疫苗的水分含量分别为1.7％、2.0％和2.1％,疫苗的A、C、W135、Y群脑膜炎球菌多糖含量和回收率分别都≥50 μg/剂和80％,符合相关规定的要求.结论 按确定的工艺成功制备ACYW135群脑膜炎球菌多糖疫苗.     

A、C、Y和W135群四价脑膜炎球菌多糖菌苗的临床和血清学评价

许多急性脑膜炎球菌感染是由 Y 和W135群脑膜炎球菌引起的,为了扩大脑膜炎菌苗的保护效果,应将 Y 和 W135群多糖加到A 和 C 群多糖中。本研究旨在评价这种四价制剂临床上的     

脑膜炎球菌病的流行病学及疫苗(待续)

脑膜炎球菌是唯一能导致全球流行性脑脊髓膜炎(流脑)流行的病因.全球范围内90％的病例是由A、B、C、Y和W135群所致.欧美国家主要流行菌群为B、C和Y群;亚洲和非洲主要是A、C和W135群.A、C、Y和W135群多糖疫苗能有效控制流脑暴发,但由于在婴儿中免疫原性差,应用并不广泛.发达国家在引进C群结合疫苗后,C群流脑发病数迅速下降.A、C、Y和W135群结合疫苗的研制成功有望控制这些群导致的流脑流行.以外膜囊、外膜蛋白和基因组来源的表面蛋白为目标的B群疫苗研究取得不同程度的进展.     

脑膜炎球菌病的流行病学及疫苗(续)

脑膜炎球菌是唯一能导致全球流行性脑脊髓膜炎(流脑)流行的病因.全球范围内90％的病例是由A、B、C、Y和W135群所致.欧美国家主要流行菌群为B、C和Y群;亚洲和非洲主要是A、C和W135群.A、C、Y和W135群多糖疫苗能有效控制流脑暴发,但由于在婴儿中免疫原性差,应用并不广泛.发达国家在引进C群结合疫苗后,C群流脑发病数迅速下降.A、C、Y和W135群结合疫苗的研制成功有望控制这些群导致的流脑流行.以外膜囊、外膜蛋白和基因组来源的表面蛋白为目标的B群疫苗研究取得不同程度的进展.     

A、C、Y 和 W_(135)群脑膜炎球菌多糖菌苗的临床及血清学评价

为了进一步扩大菌苗的保护力,作者把W_(135)群多糖加到 A、C 和 Y 群菌苗中,以观察这种混合菌苗在临床上可被接受的程度及血清学应答。使用的菌苗是用冷酚法提取的纯化多糖,用琼脂糖4B 凝胶层析法测定分子量,各种多糖均为高分子量。在分配系数0.4以前,     

Development of new vaccines against meningococcal disease

Meningococcal diseases continue to have a major public health impact in many countries. Five major groups of Neisseria meningitidis (A, B, C, Y and W135) are responsible for most meningoccocal diseases. Plain polysaccharides vaccines for Nelsseria meningitidis groups A, C, Y and W-135 have been in use for approximately 20 years, both to prevent invasive disease in high-risk population and to control disease outbreaks. However, these conventional meningococcal vaccines induce a relatively short-lasting T-cell independent immune response, are not effective in children under two years of age and can induce hyporesponsiveness. New meningococcal group C conjugate vaccines have since been developed, which offer solid advantages over the currently licensed plain polysaccharide vaccines. There is still no vaccine available against the serogroup B, which is a major cause of invasive disease. This report summarises the different approaches to the development of vaccines against the pathogenic meningococci.     

Meningococcal glycoconjugate vaccines

Neisseria meningitidis is a major cause of invasive bacterial infections worldwide. For this reason, efforts to control the disease have been directed at optimizing meningococcal vaccines and implementing appropriate vaccination policies. In the past, plain polysaccharide vaccines containing purified capsular polysaccharides A, C, Y and W135 were developed, but failed to protect infants, who are at greatest risk. Experience with the conjugate Haemophilus vaccine suggested that this approach might well empower meningococcal vaccines. Thus, a very efficacious vaccine against serogroup C Neisseria meningitis was optimized and has been widely used in developed nations since 1999. On the basis of epidemiological changes in the circulation of pathogenic serogroups in the United States, a quadrivalent conjugate vaccine against A, C, Y and W135 serogroups (Menactra?) has been developed and was approved by the U.S. FDA (Food and Drug Administration) in 2005. Recently, another tetravalent conjugate meningococcal vaccine (Menveo?) has been licensed and made available in the United States of America and in the European Union. Finally, in response to large epidemics caused by serogroup A meningococcus in Africa, a new, safe, immunogenic and affordable vaccine has been developed. This review highlights the evolution of conjugate meningococcal vaccines in general and discusses how this kind of vaccine can contribute to preventing meningococcal disease.     

Safety and immunogenicity of a meningococcal (Groups A, C, Y, W-135) polysaccharide diphtheria toxoid conjugate vaccine in healthy children aged 2 to 10 years in Chile

Immune responses to meningococcal conjugate (Menactra; MCV-4) and plain polysaccharide (Menomune-A/C/Y/W-135; PSV-4) vaccines against serogroups A, C, Y, and W-135 were assessed in 220 of 1037 Chilean children aged 2 to 10 years participating in a comparative safety trial. Both vaccines were generally well tolerated. Geometric mean serum bactericidal antibody (SBA) titers 28 days postvaccination were comparable in both groups for all four serogroups. Seroconversion was evident in > 97% of MCV-4 and > 90% of PSV-4 vaccinees who tested seronegative at baseline. Menactra safely induced broad and robust immune responses against serogroups A, C, Y and W-135 in this population.     

苯酚提取法对A群脑膜炎球菌多糖中细菌内毒素含量的影响

目的  研究苯酚提取法对A 群脑膜炎球菌多糖料液中细菌内毒素含量的影响。方法  用苯酚对A 群脑膜炎球菌多糖料液进行提取，比较粗制多糖不同溶解倍数、苯酚提取过程中采用不同搅拌转数、提取次数、提取温度等因素对多糖细菌内毒素含量的影响。  结果  粗制多糖采用4.5%醋酸钠溶液1∶100（质量体积比）溶解后进行苯酚提取的多糖细菌内毒素含量最低。同时，综合考虑多糖料液中蛋白含量、多糖回收率的检定结果，确定用苯酚进行提取的最佳条件为搅拌转数为400 转/min，提取次数为3次，温度为13～15 ℃，可有效降低多糖料液中细菌内毒素的含量。  结论  苯酚提取法可明显降低A 群脑膜炎球菌粗制多糖料液中的细菌内毒素含量。     

测定b型流感嗜血杆菌结合疫苗游离多糖的脱氧胆酸钠沉淀法的建立

 目的  建立测定b型流感嗜血杆菌（Haemophilus influenzae type b,Hib）结合疫苗游离多糖的脱氧胆酸钠（sodium deoxycholate,DOC）沉淀法。 方法   在一定的酸性条件下,用1%DOC沉淀分离Hib结合疫苗中的结合多糖和游离多糖,测定上清和沉淀的多糖含量,并对该法进行验证。 结果   DOC沉淀法的标准曲线具有可靠的线性,决定系数＞0.999。该法的准确性和精密度良好,多糖加样回收率为103%~108%,相对标准差均<10%。 结论   建立的DOC沉淀法可用于Hib结合疫苗中的游离多糖测定。     

Meningococcal disease in international travel: vaccine strategies

International travel and migration facilitate the rapid intercontinental spread of meningococcal disease. Serogroup A, and to a lesser extent serogroup C, have been responsible for pandemics in the past (mainly in Africa), but in recent years there was an international outbreak due to W135 related to the Hajj pilgrimage. The high carriage rates, persistence and transmissibility, in combination with the high case fatality rate of the Hajj-associated W135 outbreak clone, certainly raise considerable concern about the public health consequences of widespread dissemination of this organism and the potential for future epidemics. Indeed, the now evolving W135 epidemic in Africa mandates that the bivalent meningococcal vaccine should be replaced by the tetravalent meningococcal vaccine, covering A, C, Y and W135 serogroups. The currently available polysaccharide tetravalent meningococcal vaccine, albeit associated with high seroconversion and efficacy rates, has several shortcomings: it is not immunogenic in young children, duration of protective immunity is short, and it has minimal or no effect on nasopharyngeal carriage and therefore transmission of the organism. Immunogenicity of polysaccharide vaccines can be improved by chemical conjugation to a protein carrier, thereby eliciting a T-cell-dependent antibody response. In contrast to polysaccharide vaccines, conjugate vaccines are immunogenic in young infants, induce long-term protection, and reduce nasopharyngeal carriage. The tetravalent conjugate vaccine will be a leap forward in the control of meningococcal epidemics in affected countries. It will also boost the uptake of meningococcal vaccines in travelers, because the duration of protection is longer and it eliminates the problem of immune hyporesponsiveness of serogroup C with repeated dosing. The small risk of travel-associated disease for the general traveler and the unpredictable nature of epidemics make it difficult to provide evidence-based vaccine recommendations. The current recommendation is to vaccinate all Hajj pilgrims, travelers to areas with current outbreaks, travelers to the sub-Saharan meningitis belt, and high-risk individuals (i.e., those with immunodeficiencies).