A+C群脑膜炎球菌多糖疫苗稳定性研究

为确定A C群脑膜炎球菌多糖疫苗有效期。对该疫苗在不同储藏温度下的稳定性进行了系统研究,将C群多糖抗原放置在37℃,A C群多糖疫苗分别放置在2-8℃,37℃和56℃,定期取样,用琼脂糖CL-4B凝胶柱层析后,测定Kd值在0.5以前的多糖回收率,结果表明,C群多糖抗原在37℃保存9周,A C群多糖疫苗于2-8℃保存4年,37℃保存88周,56℃保存10个月,多糖抗原或多糖疫苗的Kd值小于0.4,多糖回收率大于75%,符合规程要求;因而A C群脑膜炎球菌多糖疫苗的有效期可定为在2-8℃储藏条件下3年6个月。     

B群脑膜炎球菌多组分疫苗研究进展

目前获准上市的流脑疫苗主要有A群、A+C群脑膜炎球菌疫苗及A,C,W-135及Y群的四价脑膜炎球菌疫苗,B群脑膜炎球菌疫苗尚未研制成功。近年来,研究人员以疫苗外膜蛋白为基础,应用反向疫苗学技术对B群脑膜炎球菌疫苗进行了大量研究,现重点对B群脑膜炎球菌多组分疫苗的研究进展予以综述。     

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。     

HPLC法测定A群C群脑膜炎球菌多糖疫苗中乳糖含量

建立了高效液相色谱法测定A群C群脑膜炎球菌多糖疫苗中乳糖的含量。样品经离心除去多糖后,采用阳离子交换柱分离,外标法定量分析。该法线性相关系数大于0.9999,回收率为98.8%,成品测定的CV值为1.2%(<2%),该法定量准确,重复性好,适于对A群C群脑膜炎球菌多糖疫苗中乳糖含量进行快速检测。     

A＋C群脑膜炎球菌多糖疫苗的制备及其接种人体后的血清学效果

在现行的A群脑膜料球菌多糖原液制造工艺的基础上,经部分改进后进行C群脑膜炎球菌多糖原液的生产。3批中试A＋C群脑膜炎球菌多糖疫苗经全面检定后,各项指标均符合WHO《生物制品规程》的要求。该疫苗在接种人体后,5－13岁儿童组,抗A群及抗C群脑膜炎球菌的血清杀菌抗体4倍增长率为96．59％和92．15％;≤2岁儿童组,抗A群及抗C群脑膜炎球菌的血清杀菌抗体4倍增长率为68．60％和69．77％。2组儿童接种后1个月的抗A群及抗C群膜炎球菌血清杀菌抗体几何平均滴度（GMT）与接种前均有显著性差异（P＜0．001）。     

ACYW135群脑膜炎球菌多糖疫苗的免疫原性评价

目的评价ACYW135群脑膜炎球菌多糖疫苗在昆明健康人群接种的免疫原性,为流脑防治策略提供依据。方法 2010年对昆明市2岁!3、岁!、4岁!、5岁!、6岁!、10岁!、≥15岁共7个年龄组分层随机抽取筛选出654名健康人,分别采集免前和免后1个月血清。用微量杀菌力试验(TTC法)分别检测血清中抗A、C、Y和W135群脑膜炎球菌杀菌抗体的水平。结果免后1个月抗A、C、Y和W135群脑膜炎球菌的杀菌抗体阳转率分别为96.99%、96.37%、88.43%和87.07%,抗A、C、Y和W135群膜炎球菌血清的杀菌抗体几何平均滴度(GMT)分别为1∶297.991、∶195.80、1∶72.74和1∶45.95。结论 ACYW135群脑膜炎球菌多糖疫苗在≥2岁以上的健康人群中有较好的免疫原性。     

A群C群脑膜炎球菌多糖疫苗及b型流感嗜血杆菌结合疫苗中丙酮残留量检测方法的建立与验证

目的建立A群C群脑膜炎球菌多糖疫苗和b型流感嗜血杆菌结合疫苗中丙酮残留量的检测方法并加以验证。方法参照《中华人民共和国药典》三部(2010年版)中"毛细管柱顶空进样等温法",优化色谱条件,建立A群C群脑膜炎球菌多糖疫苗和b型流感嗜血杆菌结合疫苗中丙酮残留量的检测方法并对该方法进行验证及初步应用。结果色谱条件为顶空平衡温度70℃,顶空平衡时间40 min,汽化室温度200℃,柱箱温度40℃,检测器温度250℃,进样量为1.0 m L,载气(高纯氮气)流量1.3 m L/min,尾吹气(高纯氮气)流量5 m L/min,分流比1∶1。丙酮质量分数在2×10-6~5×10-5范围内具有良好的线性关系(r0.99)。丙酮的平均回收率及相对标准偏差(RSD)分别为86.05%~105.11%及2.1%~9.5%,检测限为2×10-6,定量限为3×10-6。结论本方法的线性、特异性、准确性、重复性等均符合规定,方法准确、稳定,可用于A群C群脑膜炎球菌多糖疫苗和b型流感嗜血杆菌结合疫苗中丙酮残留量的检测。     

A群C群脑膜炎球菌结合疫苗稳定性

目的评价A群C群脑膜炎球菌结合疫苗原液和成品的稳定性。方法分别将A群、C群脑膜炎球菌结合疫苗原液及A群C群脑膜炎球菌结合疫苗各选取连续3批,分别放置于37℃、20~25℃和2~8℃3种温度下,在一定的时间取样进行主要项目测定,在关键时间点进行全面检测。结果 A群结合疫苗原液于2~8℃保存9个月,20~25℃保存4周,37℃保存4 d;C群结合疫苗原液于2~8℃保存9个月,20~25℃保存6个月,37℃保存4周;A群C群脑膜炎球菌结合疫苗于2~8℃保存2年3个月,20~25℃保存6个月,37℃可以保存9周;各项检测指标均符合质量标准的要求。结论在2~8℃条件下,A群、C群脑膜炎球菌结合疫苗原液存放6个月,A群C群脑膜炎球菌结合疫苗存放2年,其质量稳定。     

B群脑膜炎球菌疫苗的研制策略

脑膜炎奈瑟菌主要引起儿童细菌性脑脊髓膜炎和败血症,有较高的发病率和病死率。现用疫苗能够控制A、C、W135和Y群脑膜炎球菌引起的感染,而由于B群荚膜多糖免疫原性弱,外膜蛋白变异性高等原因,仍无安全和具有广泛保护性的疫苗用于控制B群脑膜炎球菌的感染。目前,B群脑膜炎球菌大多已成为引起发达国家侵袭性脑膜炎疾病的主要病原体。随着研究的不断深入,B群脑膜炎球菌疫苗的研究已经取得了很大的进展,外膜囊(Out membrane vesicles,OMV)疫苗已经在控制特异性菌株爆发流行中取得了成功。然而,人们对具有广泛保护性的B群脑膜炎球菌疫苗的探索仍在继续。本文对近年来B群脑膜炎球菌基于不同型抗原疫苗的各种研制策略及其存在的问题进行了综述。     

A+W群脑膜炎球菌外膜囊泡疫苗的临床前免疫原性和功能活性研究及其和现有疫苗的比较

<正>脑膜炎球菌A群和W群是撒哈拉沙漠以南非洲地区细菌性脑膜炎疾病流行暴发的主要原因。在本研究中,我们由非洲脑膜炎球菌A群和W群的代表性菌株制备得到了A+W群脑膜炎球菌外膜囊泡(d OMV)疫苗,并在小鼠体内比较了这种疫苗和现有的脑膜炎球菌多糖疫苗及结合疫苗的免疫原性。使用临床前批次的A+W群d OMV疫苗以及商业化应用的疫苗对NMRI小鼠进行免疫,这些疫     

The quantitative immunochemical determination of pneumococcal and meningococcal capsular polysaccharides by light scattering rate nephelometry

A quantitative nephelometric method was used for the measurement of the individual pneumococcal, as well as meningococcal, polysaccharides in the polyvalent vaccine final containers. This method is simple, rapid, inexpensive, and provides both qualitative and quantitative analyses of the polyvalent polysaccharide vaccines. By this method the individual pneumococcal types, 1, 2, 3, 4, 6A, 7F, 8, 9N, 12F, 14, 18C, 19F, 23F and 25 polysaccharides, were found to be present at 90-114% of the manufacturer's indicated concentrations; meningococcal group A, C, Y and W135 polysaccharides were at 90-108% of the manufacturer's listed concentrations. This nephelometric method coupled with gel filtration can also be used for measurement of the molecular sizes or stability of individual polysaccharides in the final container. Pneumococcal polysaccharide types 3, 6A, 9N and 19F, used as representative types, were treated with 0.5 N hydrochloric acid. The molecular sizes for types 3 and 9 N polysaccharides were stable to acid treatment. In contrast, types 6A and 19F polysaccharides were degraded. Heating meningococcal groups A, C, Y and W135 polysaccharides at 37 degrees C for 48 h did not affect their molecular size in the polyvalent vaccine.     

免疫沉淀法用于A群流脑多糖菌苗的鉴别试验

本文将免疫沉淀法——免疫双扩散及对流免疫电泳法用于Ａ群流脑多糖菌苗的鉴别试验。并对其检测Ａ群流脑多糖菌苗的特异性及敏感性与传统采用的间接血凝抑制试验法进行了比较。试验结果表明,免疫沉淀法准确、特异性好、操作简便、经济。完全符合ＷＨＯ规程及中国规程中对《Ａ群脑膜炎球菌多糖菌苗制造及检定规程》鉴别试验的要求。因此可望替代现行的间接血凝抑制试验,以作为今后Ａ群流脑多糖菌苗鉴别试验的常规测定法     

A comparative study of the immunogenicity of 2 group-B meningococcal vaccines in monkeys

The comparative study of two group B meningococcal vaccines manufactured in the USSR and in Cuba was made. The vaccine manufactured in the USSR contained the noncovalent compound of group B Neisseria meningitidis polysaccharide and outer membrane protein, and the Cuban vaccine contained group B N. meningitidis outer membrane proteins and group C N. meningitidis polysaccharide. The data obtained in this study indicated that both vaccines possessed immunological potency evaluated according to their capacity to stimulate the formation of bactericidal antibodies, whose level was found to increase eightfold after the immunization of monkeys in two injections. Besides, group B meningococcal vaccines did not induce the suppression of nonspecific protective activity characteristics of the body and did not stimulate the formation of autoantibodies to brain and liver tissues, which was indicative of the safety of these vaccines.     

冻干A+C群脑膜炎球菌多糖疫苗安全性和免疫原性观察

为了评价冻干A+C群脑膜炎球菌多糖疫苗(MPV)的安全性和免疫原性,按随机、盲法的原则,评价疫苗免疫后不良反应发生率、抗体阳转率及抗体几何平均滴度(GMT)。将606名健康观察者分为2～5岁、6～12岁、13～17岁、18～60岁四个年龄组,结果显示,该疫苗全身反应发生率2.64%,局部反应发生率1.32%,四个年龄组疫苗免疫后杀菌抗体阳性率分别为100%、100%、99.3%和100%,免疫后A群杀菌抗体GMT分别为1:250.47、1:190.61、1:144.22和1:205.79,免疫后C群杀菌抗体GMT分别为1:273.33、1:551.18、1:788.26和1:809.81,冻干A+C群多糖疫苗在≥2岁人群中具有良好的安全性和免疫原性,建议推广使用A+C群MPV。     

Meningococcal polysaccharide vaccines: A review

Polysaccharides produced by Neisseria meningitidis are pharmaceutically important molecules, and are the active components of vaccines against N. meningitidis serogroups A, C, W135 and Y. Effective vaccines based on capsular polysaccharide, polysaccharide conjugates and outer membrane vesicles have been developed for strains expressing capsular polysaccharides that define the sero groups A, C, Y and W135. However, conventional approaches to develop a vaccine for group B strains have been largely unsuccessful. This review focuses on the various aspects of fermentative production of meningococcal polysaccharide from N. meningitidis, methods of conjugation for improving the immunogenicity of polysaccharide vaccine, and efficient and cost effective methods for the purification of N. meningitidis capsular polysaccharide and outer membrane vesicles. In addition, different analytical techniques for the quantitative determination of polysaccharide vaccine and evaluation of structural integrity of conjugate vaccine have been described.     

Evidence for familial immune defect in meningococcal meningitis.

Twenty-six patients who had recovered from group A meningococcal meningitis were vaccinated with group C meningococcal polysaccharide and tetanus toxoid. Their haemagglutinating antibody response was measured two weeks later and compared with those of 22 siblings and 39 controls. Patients and siblings had a significantly lower antibody response to the group C vaccine but not to tetanus toxoid. This suggests that patients susceptible to meningococcal disease may have an immune defect involving their response to meningococcal polysaccharides.     

The effect of vaccination on local immunity indices; the determination of antimeningococcal antibodies in saliva]

Local immunity characteristics were studied in 130 young males; of these, 80 had been immunized with group A meningococcal vaccine. In nonstimulated saliva, collected prior to vaccination, then on days 7, 14 and 30 after vaccination, the levels of IgA antibodies to group A meningococcal group-specific polysaccharide (PS-A) were determined in the enzyme immunoassay, and secretory IgA and IgA, IgG, IgM were determined by Mancini's method. The study revealed that after the parenteral administration of group A meningococcal vaccine an increase in the concentrations of SIgA and IgA antibodies to PS-A occurred. The manifestation of changes in local immunity characteristics in response to meningococcal vaccine depended on the initial level of IgA antibodies to PS-A.     

Purification of capsular polysaccharide from Neisseria meningitidis serogroup C by liquid chromatography

Neisseria meningitidis serogroup C capsular polysaccharide (MenCPS) is an important antigen against meningococcal infection. This paper describes a new purification methodology employing liquid chromatography that resulted in a polysaccharide showing the characteristics recommended by the World Health Organization for vaccine purposes. In this method, steps of the traditional procedure that yield low recovery and use toxic materials were modified. The present process consists in the following steps: (1) continuous flow centrifugation of the culture for removal of the cells; (2) supernatant concentration by tangential filtration (100 kDa cutoff); (3) addition of 0.5% DOC, heating to 55 degrees C during 30 min and tangential filtration (100 kDa cutoff); (4) anion exchange chromatography (Source 15Q) and (5) size exclusion chromatography (Sepharose CL-4B). The polysaccharide C fraction obtained in that way was dialyzed and freeze-dried. The structural identity of the polysaccharide was demonstrated by (1)H-NMR spectrometry.     

Simple and rapid technique for monitoring the quality of meningococcal polysaccharides by high performance size-exclusion chromatography

The molecular size of meningococcal polysaccharides is an important physico-chemical parameter which correlates with immunogenicity. This paper describes the experimental conditions for high-performance size-exclusion chromatography on a PL Aquagel-OH 60 column to follow changes in the size distribution and therefore in the distribution coefficient (K(D)) of the meningococcal polysaccharides of groups A, C, Y and W-135 used to formulate anti-Neisseria meningitidis vaccines. The experimental conditions were also found to be suitable for a rapid monitoring of the quality (no group A polysaccharide depolymerization) of the tetravalent meningococcal polysaccharide vaccine.