Developing a novel multiplex assay using Luminex xMAP technology

鉴定出与肺炎球菌感染保护相关的非型别依赖性细胞和体液相关物将加速具有广泛保护作用的疫苗的开发。使用实验性人类肺炎球菌挑战（EHPC）模型样本生物库和纯化的肺炎球菌蛋白的大型文库来开发多重测定以测量针对肺炎球菌（Spn）的全身体液蛋白特异性应答。开发并优化了多重Luminex xMAP测定。来自鼻内接种Spn6B和Spn15B的EHPC志愿者的血清样品用于测定验证，并测量了用细菌实验性激发的志愿者对74种肺炎球菌蛋白抗原的体液应答。使用中值荧光强度值报告抗蛋白IgG水平。易受Spn6B实验性定殖的志愿者在激发后29天与激发前相比引起对各种抗原的抗体水平增加。在受保护免受殖民的志愿者中没有检测到这种趋势。基线时，易感队列和受保护队列之间的差异极小。与Spn6B诱导的应答相比，Spn15 B实验定殖后的体液应答降低，表明Spn15 B的免疫原性有限。因此，开发了一种新型的多重测定法，可以快速、经济有效地测定对大型肺炎球菌候选疫苗库的蛋白质特异性抗体反应。

The identification of serotype-independent cellular and humoral correlates of protection against pneumococcal infection will accelerate development of vaccines with broad protection. The Experimental Human Pneumococcal Challenge (EHPC) model biobank of samples and a large library of purified pneumococcal proteins were used to develop a multiplex assay to measure systemic humoral protein-specific responses against pneumococcus (Spn). A multiplex Luminex xMAP assay was developed and optimised. Serum samples from EHPC volunteers intranasally inoculated with Spn6B and Spn15B were used for assay validation and humoral response to 74 pneumococcal protein antigens in volunteers experimentally challenged with the bacteria was measured. Median fluorescence intensity values were used to report anti-protein IgG levels. Volunteers susceptible to experimental colonisation with Spn6B elicited increased antibody levels to various antigens 29 days post-challenge compared to pre-challenge. This trend was not detected in volunteers who were protected against colonisation. Minimal difference was identified between susceptible and protected cohorts at baseline. Humoral response following experimental colonisation with Spn15B was reduced in comparison to response induced by Spn6B, suggesting limited immunogenicity of Spn15B. A novel multiplex assay has thus been developed allowing rapid, cost-effective determination of protein-specific antibody responses to a large library of pneumococcal vaccine candidates.