Polymer adjuvants for innate and cellular based vaccination

在商业疫苗生产的早期，同一疫苗不同批次的有效性之间的显著差异被正确地归因于反应容器的污染。然而，更加注重清洁似乎降低了疫苗的有效性，这表明污染物实际上增强了免疫原性。这些污染物被称为“佐剂”；刺激免疫系统并增加对疫苗反应的药剂，但没有任何内在的抗原性作用。他们通过创造一个非特异性的促炎环境来做到这一点，在这个环境中，特定抗原的呈现导致疫苗反应的效率要高得多。经验表明，佐剂对研制有效疫苗起着至关重要的作用，许多佐剂现已广泛使用，包括油、铝盐和病毒体。疫苗接种领域的重要性日益增加，从1977年成功消灭天花（估计挽救了3000万人的生命），到迫切需要针对艾滋病毒、疟疾、结核病和癌症的预防性（最好是治疗性）疫苗。新佐剂将对新疫苗的开发做出重要贡献，因为迄今为止一些最有效的佐剂（如Freund佐剂）不能按照cGMP生产，不适合临床使用。解决这一不足的一种方法是设计和生产适用于cGMP生产的强效和特异性合成分子佐剂。我们将合成聚合物- tlr配体缀合物，它能够结合它们的受体并激活程序化的细胞因子反应。多价反应性聚合物将采用可控自由基聚合的方法合成，以获得分子量分布较窄的聚合物。亲水单体，如聚乙二醇甲基丙烯酸酯或N，（2-羟丙基甲基丙烯酰胺）将与活性酯承载单体或叠氮化物承载单体共聚合，用于随后的改性。为了确定材料诱导免疫反应的最佳能力，将检查聚合物结构的三个参数：首先是聚合物的分子量和多分散性；其次是每个聚合物中TLR配体的数量，最后是间隔层的长度和性质。所有这些参数将并行调整，并且对于理解材料的特征如何改变免疫反应非常重要。生物活性将通过报告细胞进行初步评估，报告细胞在任何TLR配体的刺激下被激活。报告蛋白被表达并反映细胞的激活水平，因此是刺激的指标。其次，通过测量激活后释放的细胞因子的相对数量，将评估配体-聚合物偶联物的特定活性，以确定该材料是否刺激基于细胞或基于抗体的免疫反应。

In the early days of commercial vaccine manufacture, significant variation between the effectiveness of different batches of the same vaccine were correctly ascribed to contamination of the reaction vessels. However, more scrupulous attention to cleanliness seemed to reduce the effectiveness of the vaccines, suggesting the contaminants actually enhanced immunogenicity. These contaminants were described as 'adjuvants'; agents which stimulate the immune system and increase the response to a vaccine, without having any intrinsic antigenic effect. They do this by creating a non-specific pro-inflammatory environment, where presentation of specific antigens leading to a vaccine response is much more efficient. Experience shows that adjuvants make a crucial contribution to the development of effective vaccines, and many adjuvants are now in widespread use, including oils, aluminium salts and virosomes.The field of vaccination is of ever-increasing importance, ranging from successful eradication of Smallpox in 1977 (estimated to have saved 30 m lives) through to the pressing need for prophylactic (and ideally therapeutic) vaccines for HIV, malaria, TB and cancer. New adjuvants will make an important contribution to the development of new vaccines, as some of the most effective adjuvants to date (such as Freund's adjuvants) cannot be manufactured to cGMP and are not suitable for clinical use. One way to address this shortfall is to design and produce potent and specific synthetic molecular adjuvants which are suitable for cGMP manufacture.We will synthesize polymer-TLR ligands conjugates which are capable of binding their receptor and activate a programmed cytokine response. Multivalent reactive polymers will be synthesized using controlled radical polymerization to obtain polymers which have a narrow molecular weight distribution. Hydrophilic monomers such as polyethylene glycol methacrylate, or N,(2-hydroxypropylmethacrylamide) will be co-polymerized with either an activated ester bearing monomer or an azide bearing monomer for subsequent modification. Three parameters of polymer structure will be examined in order to determine optimum capacity of the materials to induce an immune response: Firstly the molecular weight and polydispersity of the polymer; secondly the number of TLR ligands per polymer and finally the spacer length and properties. All of these parameters will be adjusted in parallel and are important in understanding how features of the material alter the immune response.Biological activity will be initially assessed using reporter cells which are activated upon stimulation of any TLR ligands. A reporter protein is expressed and reflects the level of activation of the cells and thus an indicator of stimulation. Secondly, the specific activity of the ligand-polymer conjugates will be assessed to determine whether the materials stimulate either a cell based or antibody based immune response by measuring the relative quantities of the cytokines released following activation.