Polysaccharide and conjugate vaccine quality control

多糖和结合疫苗的质量控制

• 批准号: 6838975
• 负责人: CHI-JEN LEE
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6838975
• 关键词: CpG islands; Streptococcus pneumoniae; active immunization; bacterial polysaccharides; drug quality /standard; glycolipids; glycoproteins; immunoconjugates; immunomodulators; laboratory mouse; microorganism immunology; mucosal immunity; phagocytosis; vaccine development

Pneumococci continue to be the major cause of pneumonia, meningitis, and otitis media in young children and other high risk population worldwide. Pneumococcal polysaccharide (PS), designated as T-cell independent type 2 (TI-2) antigen, induces poor immune response in young children. Splenic marginal zone B cells, associated with CD21, CD19 and C3d, play an important role in TI-2 antibody responses, and provide host defense against bacterial pathogens. We have studied the antibody response, avidity, and opsonophagocytic activity of antisera in mice immunized with type 9V PS conjugated to inactivated pneumolysin (Ply) or autolysin (Aly), and 7-valent pneumococcal conjugate vaccine. Compared to mice given 9V PS alone, serum IgG and IgM concentrations against the 9V PS were higher in mice immunized with conjugates. High concentrations of serum antibodies were maintained for over 12 weeks. The relative avidities of IgG and IgM antibodies and opsonophagocytic activity against 9V pneumococci were high in mice immunized with conjugates. Thus, conjugate vaccines can induce high as well as long duration of antibody response and effective functional activity. In another study, mice received intranasal immunization with type 9V conjugate or 9V PS. These animals produced high concentrations of 9V PS IgG and IgA antibodies in their serum, spleen, intestine, lung, Peyer's patch and fecal extract samples. Mice immunized with these glycoconjugates exhibited opsonophagocytic activity and rapid bacterial clearance from blood and provided homologous and cross-protection against challenge with virulent pneumococci. These results indicate that intranasal immunization with glycoconjugate vaccines may serve as an alternative and convenient approach for prevention of pneumococcal infection. Most antigens are poor mucosal immunogens and need effective adjuvants. Mucosal immune responses against pneumococcal conjugates have been conducted using CpG oligodinucleotide (ODN). The adjuvant activity of CpG ODN may associate with the binding of CpG to the Toll-like receptor 9, induction of Th1-inducing cytokines, MHC and costimulatory molecules of antigen presenting cells. High IgG and IgA antibody responses were observed in the samples including serum, Peyer's patch, and intestine of mice immunized intranasally with 9V PS-Ply conjugate. In contrast, no significant difference in antibody responses were observed between mice given 9V PS and non-immunized control group. These results suggest that CpG could serve as an effective adjuvant to induce mucosal immunity for pneumococcal conjugate vaccine. Different bacterial vaccines including Haemophilus b conjugate, pneumococcal type 6 B PS, and typhoid Vi PS vaccines were irradiated with various doses of gamma, electron beam and X-ray. In general, there is correlation between the irradiation dosage and increased values of the Kd, and therefore, decreased the molecular size and stability of these vaccines. Quality control of pneumococcal Polysaccharide-protein conjugate vaccines. Protection against pneumococcal infection depends on the presence of antibodies against capsular polysaccharides (PSs). These serotype-specific antibodies facilitate phagocytosis to destroy invading bacteria. The spleen is an important organ in the immune response to pneumococci, since it contains both antibody-producing B-cells and phagocytes. The 23-valent pneumococcal PS vaccine is effective against bacteremic pneumococcal disease in adults and is cost-effective for routine immunization of the elderly. However, the PS vaccine is not sufficiently immunogenic in infants and young children. Chemical coupling of a PS to a carrier protein to form a glycoconjugate greatly improves the immunogenicity of a PS. The 7-valent pneumococcal conjugate vaccine, Prevnar, appears to be highly effective in preventing invasive disease in young children and to have a significant impact on otitis media. Studies were conducted in our laboratory to evaluate the avidity, antibody response and opsonophagocytic activity of serum antibodies to the pneumococcal type 9V PS conjugated to inactivated pneumolysin (Ply) or autolysin (Aly) and in the 7-valent PS-CRM197 conjugate vaccine. The effect of priming mice with the 7-valent conjugate followed by a booster dose of 23-valent PS vaccine was also examined. Compared to 9V PS alone or non-immunized controls, the serum concentrations of 9V PS IgG and IgM antibodies were significantly higher in mice immunized with 9V PS-Ply, 9V PS-Aly conjugates, or 7-valent conjugate vaccine at 0, 2, and 4 weeks, 7-valent conjugate followed by 23-valent PS vaccine compared to 9V PS alone. Thus, priming mice with polyvalent conjugate vaccine induced high 9V PS antibody response in contrast to the group primed with polyvalent PS vaccine. The duration of antibody response in mice was studied in mice immunized with various conjugate vaccines and boosted with 23-valent PS vaccine. The antibody response was highest at 4 weeks after the injection, increasing 7-10 fold compared to the group that received 9V PS alone. The high IgG Ab response was maintained for over 12 weeks after final injection. Similar results were also observed in 9V PS IgM Ab response. In addition, high IgG and IgM Abs were maintained for over 8 weeks after final injection in mice given a booster dose of 23-valent PS vaccine. The avidity indeces of IgG antibody were highest in mice immunized twice with the 7-valent conjugate vaccine, and combined immunization with 7-valent conjugate then 23-valent PS vaccine, followed by monovalent 9V PS-protein conjugates. Mice immunized with 9V PS alone exhibited the lowest avidity. Similar results were observed in the avidity of IgM antibody. The 9V PS-Ply and 9V PS-Aly immunized group showed the opsonophagocytic titers of 16 compared to 4 in 9V PS-immunized and complement control groups. Mice immunized twice with 7-valent conjugate or conjugate vaccine followed by 23-valent PS vaccine showed titers of 32 and >64 respectively. These results indicate that monovalent and 7-valent conjugate vaccines can stimulate effective functional activity that correlated with protective immunity against pneumococcal infection. Pathogenic pneumococci enter the body through the respiratory mucosa and may cause serious infections such as pneumonia, bacteremia, and meningitis in high risk population. They are also a common cause of mucosal infection, e.g. otitis media and sinusitis in young children. The potential of mucosal immunity to protect against pneumococcal infection was studied in mice through intranasal immunization using pneumococcal type 9V PS and 9V glycoconjugates. Compared to the non-immunized control group, concentrations of 9V IgG and IgA antibodies in serum, spleen, intestine, lung, Peyer's patch and fecal extract samples were significantly higher in mice immunized with 9V PS-Ply, or 9V PS-Aly, as well as 9V PS alone. The opsonophagocytic titers in serum and intestine homogenate samples were significantly higher in the immunized groups compared to the complement control group. Furthermore, the mice immunized with 9V PS-Ply, 9V PS, or 7-valent conjugate vaccine showed more rapid bacterial clearance from blood at 1, 3, and 5 hrs after challenge with 9V pneumococci compared to the non-immunized control group. Mice immunized with 9V PS-Ply, or 7-valent conjugate vaccine also showed more rapid bacterial clearance after challenge with 19F pneumococci compared to the non-immunized control group. These functional data thus indicate that mice immunized with PS-protein conjugate vaccines had high serum opsonophagocytic activity, rapid bacterial clearance from blood, and provided cross- protection against tested heterologous pneumococcal serotype. Most antigens are poor mucosal immunogens and need effective adjuvants. A number of mucosal adjuvants have been proposed, including bacterial toxin, such as cholera toxin and E. coli heat-labile enterotoxin, and CpG oligodeoxydinucleotide (ODN), In present study, CpG ODN was used as adjuvant. High IgG and IgA antibody responses were observed in the samples including serum, Peyer's patch, and intestine of mice immunized intranasally with 9V PS-Ply conjugate. In contrast, no significant difference in antibody responses were observed between mice given 9V PS and non-immunized control group. These results suggests that CpG could serve as an effective adjuvant to induce mucosal immunity for pneumococcal conjugate vaccine. Gamma radiation has been used for the terminal sterilization and viral inactivation of the biological products. In present study, different bacterial vaccines including Haemophilus b conjugate, pneumococcal type 6B PS, and typhoid Vi PS vaccines were irradiated with various doses of gamma, electron beam and X-ray. In general, there is correlation between the irradiation dosage and increased values of the Kd, and therefore, decreased the molecular size and stability of these vaccines.

肺炎球菌仍然是全世界幼儿和其他高危人群肺炎、脑膜炎和中耳炎的主要原因。 肺炎球菌多糖 (PS) 被称为 T 细胞非依赖性 2 型 (TI-2) 抗原，会导致幼儿免疫反应较差。 脾边缘区 B 细胞与 CD21、CD19 和 C3d 相关，在 TI-2 抗体反应中发挥重要作用，并提供宿主针对细菌病原体的防御。 我们研究了用灭活肺炎球菌溶血素 (Ply) 或自溶素 (Aly) 缀合的 9V PS 和 7 价肺炎球菌缀合疫苗免疫小鼠的抗血清的抗体反应、亲合力和调理吞噬活性。 与单独给予 9V PS 的小鼠相比，用缀合物免疫的小鼠中针对 9V PS 的血清 IgG 和 IgM 浓度较高。 高浓度的血清抗体可维持超过 12 周。 在用缀合物免疫的小鼠中，IgG 和 IgM 抗体的相对亲合力以及针对 9V 肺炎球菌的调理吞噬活性较高。 因此，结合疫苗可以诱导高且持续时间长的抗体反应和有效的功能活性。 在另一项研究中，小鼠接受了 9V 型缀合物或 9V PS 的鼻内免疫。 这些动物在其血清、脾脏、肠、肺、集合淋巴结和粪便提取物样本中产生高浓度的 9V PS IgG 和 IgA 抗体。 用这些糖复合物免疫的小鼠表现出调理吞噬活性和快速从血液中清除细菌，并提供同源和交叉保护以抵抗有毒力肺炎球菌的攻击。这些结果表明，糖复合物疫苗的鼻内免疫可以作为预防肺炎球菌感染的替代且方便的方法。 大多数抗原是较差的粘膜免疫原，需要有效的佐剂。 针对肺炎球菌缀合物的粘膜免疫反应已使用 CpG 寡二核苷酸 (ODN) 进行。 CpG ODN 的佐剂活性可能与 CpG 与 Toll 样受体 9 的结合、Th1 诱导细胞因子、MHC 和抗原呈递细胞共刺激分子的诱导有关。 在用 9V PS-Ply 缀合物鼻内免疫的小鼠的血清、派尔氏集结和肠道等样品中观察到高 IgG 和 IgA 抗体反应。 相比之下，给予 9V PS 的小鼠和未免疫对照组的小鼠之间没有观察到抗体反应的显着差异。 这些结果表明，CpG 可作为诱导肺炎球菌结合疫苗粘膜免疫的有效佐剂。 用不同剂量的伽马射线、电子束和 X 射线照射不同的细菌疫苗，包括嗜血杆菌 B 结合物、肺炎球菌 6 型 B PS 疫苗和伤寒 Vi PS 疫苗。 一般来说，辐照剂量和 Kd 值增加之间存在相关性，因此，这些疫苗的分子大小和稳定性降低。 肺炎球菌多糖-蛋白结合疫苗的质量控制。 对肺炎球菌感染的保护取决于荚膜多糖 (PS) 抗体的存在。 这些血清型特异性抗体促进吞噬作用以消灭入侵的细菌。 脾脏是肺炎球菌免疫反应的重要器官，因为它含有产生抗体的 B 细胞和吞噬细胞。 23价肺炎球菌PS疫苗对成人菌血症性肺炎球菌疾病有效，对于老年人常规免疫具有成本效益。 然而，PS 疫苗对婴幼儿的免疫原性不够。 PS 与载体蛋白化学偶联形成糖缀合物，大大提高了 PS 的免疫原性。 7 价肺炎球菌结合疫苗 Prevnar 似乎对预防幼儿侵袭性疾病非常有效，并对中耳炎有显着影响。 我们实验室进行了研究，以评估与灭活肺炎球菌溶血素 (Ply) 或自溶素 (Aly) 结合的 9V 型肺炎球菌 PS 以及 7 价 PS-CRM197 结合疫苗中血清抗体的亲和力、抗体反应和调理吞噬活性。 还检查了用 7 价缀合物免疫小鼠，然后再注射加强剂量的 23 价 PS 疫苗的效果。 与单独的9V PS或未免疫对照相比，在第0、2和4周时用9V PS-Ply、9V PS-Aly缀合物或7价缀合物疫苗免疫的小鼠中9V PS IgG和IgM抗体的血清浓度显着较高，其中7价缀合物随后是23价PS疫苗与单独的9V PS相比。 因此，与用多价PS疫苗初免的组相比，用多价缀合物疫苗初免的小鼠诱导高9V PS抗体应答。 在用各种结合疫苗免疫并用 23 价 PS 疫苗加强的小鼠中研究了小鼠抗体反应的持续时间。 注射后 4 周抗体反应最高，与单独接受 9V PS 的组相比增加了 7-10 倍。 最终注射后，高 IgG Ab 反应可维持超过 12 周。 在 9V PS IgM Ab 反应中也观察到类似的结果。 此外，在给予加强剂量的 23 价 PS 疫苗的小鼠中最后一次注射后，高 IgG 和 IgM 抗体可维持超过 8 周。 7价结合疫苗免疫两次的小鼠中IgG抗体的亲合力指数最高，并且联合免疫7价结合物然后是23价PS疫苗，然后是单价9V PS-蛋白结合物。 单独使用 9V PS 免疫的小鼠表现出最低的亲合力。 在 IgM 抗体的亲和力中也观察到类似的结果。 9V PS-Ply和9V PS-Aly免疫组显示调理吞噬滴度为16，而9V PS免疫组和补体对照组显示调理吞噬滴度为4。 用7价缀合物或缀合物疫苗免疫两次，然后用23价PS疫苗免疫的小鼠分别显示出32和>64的滴度。 这些结果表明，单价和七价结合疫苗可以刺激与针对肺炎球菌感染的保护性免疫相关的有效功能活性。 致病性肺炎球菌通过呼吸道粘膜进入人体，在高危人群中可能引起肺炎、菌血症、脑膜炎等严重感染。 它们也是粘膜感染的常见原因，例如幼儿中耳炎和鼻窦炎。 通过使用肺炎球菌 9V PS 和 9V 糖缀合物进行鼻内免疫，研究了小鼠粘膜免疫预防肺炎球菌感染的潜力。 与未免疫对照组相比，用9V PS-Ply、或9V PS-Aly以及单独9V PS免疫的小鼠的血清、脾脏、肠、肺、派尔氏集结和粪便提取物样品中的9V IgG和IgA抗体浓度显着较高。 与补体对照组相比，免疫组的血清和肠匀浆样品中的调理吞噬滴度显着较高。 此外，与未免疫的对照组相比，用 9V PS-Ply、9V PS 或 7 价结合疫苗免疫的小鼠在用 9V 肺炎球菌攻击后 1、3 和 5 小时显示出更快的血液细菌清除率。 与未免疫的对照组相比，用 9V PS-Ply 或 7 价结合疫苗免疫的小鼠在用 19F 肺炎球菌攻击后也表现出更快的细菌清除率。 因此，这些功能数据表明，用PS-蛋白缀合物疫苗免疫的小鼠具有高血清调理吞噬活性、从血液中快速清除细菌，并提供针对测试的异源肺炎球菌血清型的交叉保护。 大多数抗原是较差的粘膜免疫原，需要有效的佐剂。 已经提出了许多粘膜佐剂，包括细菌毒素，例如霍乱毒素和大肠杆菌不耐热肠毒素，以及CpG寡脱氧二核苷酸（ODN）。在本研究中，使用CpG ODN作为佐剂。 在用 9V PS-Ply 缀合物鼻内免疫的小鼠的血清、派尔氏集结和肠道等样品中观察到高 IgG 和 IgA 抗体反应。 相比之下，给予 9V PS 的小鼠和未免疫对照组的小鼠之间没有观察到抗体反应的显着差异。 这些结果表明，CpG 可以作为诱导肺炎球菌结合疫苗粘膜免疫的有效佐剂。 伽马辐射已用于生物制品的最终灭菌和病毒灭活。 在本研究中，不同的细菌疫苗，包括嗜血杆菌b结合物、肺炎球菌6B型PS疫苗和伤寒Vi PS疫苗，用不同剂量的伽马射线、电子束和X射线照射。 一般来说，辐照剂量和 Kd 值增加之间存在相关性，因此，这些疫苗的分子大小和稳定性降低。