肺炎链球菌疫苗在防御由肺炎链球菌引发的各类侵袭性与非侵袭性疾病中扮演着不可或缺的角色,荚膜多糖作为关键抗原成分,杂质残留直接造成了肺炎多糖疫苗以及多糖结合疫苗在临床使用中的不良反应。随着全球对涵盖广泛血清型的高价肺炎链...肺炎链球菌疫苗在防御由肺炎链球菌引发的各类侵袭性与非侵袭性疾病中扮演着不可或缺的角色,荚膜多糖作为关键抗原成分,杂质残留直接造成了肺炎多糖疫苗以及多糖结合疫苗在临床使用中的不良反应。随着全球对涵盖广泛血清型的高价肺炎链球菌疫苗需求的持续增长,研发高效、高纯度且可大规模生产的荚膜多糖抗原技术愈发显得紧迫且至关重要。荚膜多糖的质量属性受到多维度因素的交互影响,涵盖了病原菌株的选择、培养基配方、发酵过程调控以及纯化技术等诸多环节。遵循质量源于设计(quality by design,QbD)的质量管理理念与工艺开发路径,本文系统性总结了从上游菌株筛选优化、精细化发酵工艺设计,直到下游纯化技术的全过程的知识与经验,旨在为构建和完善荚膜多糖生产工艺体系提供理论指导与实践策略。展开更多
Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreac...Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.展开更多
Pneumonia remains the single leading cause of childhood death worldwide.Despite the commercial availability of multiple pneumococcal conjugate vaccines(PCVs),high dosage cost and supply shortages prevent PCV delivery ...Pneumonia remains the single leading cause of childhood death worldwide.Despite the commercial availability of multiple pneumococcal conjugate vaccines(PCVs),high dosage cost and supply shortages prevent PCV delivery to much of the developing world.The current work presents high-yield pneumococcal conjugates that are immunogenic in animals and suitable for use in human vaccine development.The 13-valent pneumococcal conjugate vaccine(PCV-13)investigated in this research incorporated serotypes 1,3,4,5,6A,6B,7F,9V,14,18C,19A,19F,and 23F.Pneumococcal polysaccharides(PnPSs)and CRM197 carrier protein were produced and purified in-house,and used to prepare PnPS-CRM conjugates using unique,cyanide-free,in vacuo glycation conjugation methods.In vitro characterization confirmed the generation of higher molecular weight PnPS-CRM conjugates low in free protein.In vivo animal studies were performed to compare PnuVax's PCV-13 to the commercially available PCV-13,Prevnar®13(Pfizer,USA).A boost dose was provided to all groups post-dose 1 at t?14 days.Post-dose 2 results at t?28 days showed that all 13 serotypes in PnuVax's PCV-13 were boostable.Per serotype IgG GMCs demonstrated that PnuVax's PCV-13 is immunogenic for all 13 serotypes,with 10 of the 13 serotypes statistically the same or higher than Prevnar®13 post-dose 2.As a result,the novel polysaccharideprotein conjugates developed in this work are highly promising for use in human PCV development.The in vacuo conjugation technique applied in this work could also be readily adapted to develop many other conjugate vaccines.展开更多
文摘肺炎链球菌疫苗在防御由肺炎链球菌引发的各类侵袭性与非侵袭性疾病中扮演着不可或缺的角色,荚膜多糖作为关键抗原成分,杂质残留直接造成了肺炎多糖疫苗以及多糖结合疫苗在临床使用中的不良反应。随着全球对涵盖广泛血清型的高价肺炎链球菌疫苗需求的持续增长,研发高效、高纯度且可大规模生产的荚膜多糖抗原技术愈发显得紧迫且至关重要。荚膜多糖的质量属性受到多维度因素的交互影响,涵盖了病原菌株的选择、培养基配方、发酵过程调控以及纯化技术等诸多环节。遵循质量源于设计(quality by design,QbD)的质量管理理念与工艺开发路径,本文系统性总结了从上游菌株筛选优化、精细化发酵工艺设计,直到下游纯化技术的全过程的知识与经验,旨在为构建和完善荚膜多糖生产工艺体系提供理论指导与实践策略。
文摘Natural antibodies serve as the body’s first line of defense against pneumococcal challenge. Polyreactive human pneumococcal polysaccharide IgG antibodies have not been extensively studied. We analyzed human polyreactive antibodies that bind multiple pneumococcal polysaccharides, including PPS14 and PPS23F. These antibodies were isolated from single pneumococcal polysaccharide specific B cells allowing for the analysis of human immunoglobulins with natively paired variable regions. Although isolated individually, these antibodies demonstrated similar characteristics. Most antibodies possessed a variable light chain with a CDR3 length made up of nine amino acids and relatively high number of flexible amino acids in combined VH/VL. While these antibodies were polyreactive and structurally alike, kinetic analysis revealed unique KD values. Variable chains are responsible for antigen recognition whereas antibody fine specificity is affected by isotype structure. To investigate the contribution of the constant region of these isotypes and their effect on antibody avidity to pneumococcal polysaccharide, the polyreactive variable regions were expressed as IgG1 or IgG2 and subjected to kinetic analysis. The IgG1 antibodies uniformly had a stronger avidity to PPS14 and PPS23F compared to IgG2. To further document the importance of the constant region in antibody avidity and fine specificity, analysis of antibody F(ab)’2 fragment binding to PPS14 and PPS23F resulted in similar KD values. These studies suggest that antigen recognition by polyreactive antibodies is determined by a conserved variable light chain CDR3 length and longer, more flexible variable heavy CDR3s when compared to pneumococcal polysaccha-ride-specific sequences while differences in specific avidities are modulated by antibody isotype.
文摘Pneumonia remains the single leading cause of childhood death worldwide.Despite the commercial availability of multiple pneumococcal conjugate vaccines(PCVs),high dosage cost and supply shortages prevent PCV delivery to much of the developing world.The current work presents high-yield pneumococcal conjugates that are immunogenic in animals and suitable for use in human vaccine development.The 13-valent pneumococcal conjugate vaccine(PCV-13)investigated in this research incorporated serotypes 1,3,4,5,6A,6B,7F,9V,14,18C,19A,19F,and 23F.Pneumococcal polysaccharides(PnPSs)and CRM197 carrier protein were produced and purified in-house,and used to prepare PnPS-CRM conjugates using unique,cyanide-free,in vacuo glycation conjugation methods.In vitro characterization confirmed the generation of higher molecular weight PnPS-CRM conjugates low in free protein.In vivo animal studies were performed to compare PnuVax's PCV-13 to the commercially available PCV-13,Prevnar®13(Pfizer,USA).A boost dose was provided to all groups post-dose 1 at t?14 days.Post-dose 2 results at t?28 days showed that all 13 serotypes in PnuVax's PCV-13 were boostable.Per serotype IgG GMCs demonstrated that PnuVax's PCV-13 is immunogenic for all 13 serotypes,with 10 of the 13 serotypes statistically the same or higher than Prevnar®13 post-dose 2.As a result,the novel polysaccharideprotein conjugates developed in this work are highly promising for use in human PCV development.The in vacuo conjugation technique applied in this work could also be readily adapted to develop many other conjugate vaccines.
