The number and spacing of B-cell epitopes on antigens have a profound impact on the activation of B cells and elicitation of antibody responses, the quantitative aspects of which may be utilized for rational design of vaccines. Ni-chelating liposomes have been widely used as protein carriers in experimental studies of vaccine delivery, owing to the convenience and versatility of this conjugation chemistry. However, the epitope number per particle as well as the stability of protein conjugation on liposomes remain far less characterized. Here we have developed quantitative methods to measure the average spatial density of proteins on liposomes using both ensemble and single-molecule techniques and demonstrated their utility using liposomes conjugated with native proteins of two different sizes. These studies revealed that the initial density of protein conjugation on Ni-chelating liposomes can be finely controlled, but the density can decrease over time upon dilution due to the noncovalent nature of Ni-chelation chemistry. These results indicate that an alternative method other than the Ni-chelation chemistry is needed for stable conjugation of epitopes onto liposomes and also suggest a general strategy that can be used to precisely regulate the epitope density on liposomes for B-cell antigen delivery.

中文翻译：

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脂质体上蛋白质缀合的定量和稳定性，用于控制表面抗原决定簇的密度。

抗原上B细胞表位的数量和间隔对B细胞的活化和抗体应答的诱导具有深远的影响，其定量方面可用于疫苗的合理设计。由于该缀合化学的方便性和多功能性，镍螯合脂质体已广泛用于疫苗输送的实验研究中作为蛋白质载体。然而，每个颗粒的表位数目以及脂质体上蛋白质缀合的稳定性仍然远远不够。在这里，我们已经开发了定量方法，可以使用集成技术和单分子技术来测量脂质体上蛋白质的平均空间密度，并证明了脂质体与两种不同大小的天然蛋白质缀合后的效用。这些研究表明，可以很好地控制镍螯合脂质体上蛋白质缀合的初始密度，但由于镍螯合化学的非共价性质，其密度在稀释后会随着时间的流逝而降低。这些结果表明，除了Ni螯合化学以外，还需要其他方法来将表位稳定地缀合到脂质体上，并且还提出了可用于精确调节脂质体上表位密度以进行B细胞抗原递送的一般策略。