Novartis Vaccines and Diagnostics srl, Siena, Italy.
PLoS One. 2013 Aug 15;8(8):e70620. doi: 10.1371/journal.pone.0070620. eCollection 2013.
Understanding the impact that human memory B-cells (MBC), primed by previous infections or vaccination, exert on neutralizing antibody responses against drifted influenza hemagglutinin (HA) is key to design best protective vaccines. A major obstacle to these studies is the lack of practical tools to analyze HA-specific MBCs in human PBMCs ex vivo. We report here an efficient method to identify MBCs carrying HA-specific BCR in frozen PBMC samples. By using fluorochrome-tagged recombinant HA baits, and vaccine antigens from mismatched influenza strains to block BCR-independent binding, we developed a protocol suitable for quantitative, functional and molecular analysis of single MBCs specific for HA from up to two different influenza strains in the same tube. This approach will permit to identify the naive and MBC precursors of plasmablasts and novel MBCs appearing in the blood following infection or vaccination, thus clarifying the actual contribution of pre-existing MBCs in antibody responses against novel influenza viruses. Finally, this protocol can allow applying high throughput deep sequencing to analyze changes in the repertoire of HA⁺ B-cells in longitudinal samples from large cohorts of vaccinees and infected subjects with the ultimate goal of understanding the in vivo B-cell dynamics driving the evolution of broadly cross-protective antibody responses.
了解先前感染或接种疫苗引发的记忆 B 细胞（MBC）对漂移的流感血凝素（HA）中和抗体反应的影响，是设计最佳保护疫苗的关键。这些研究的主要障碍是缺乏实用工具来分析人类 PBMC 中的 HA 特异性 MBC。我们在这里报告了一种在冷冻 PBMC 样本中鉴定携带 HA 特异性 BCR 的 MBC 的有效方法。通过使用荧光标记的重组 HA 诱饵，以及来自不匹配流感株的疫苗抗原来阻断 BCR 非依赖性结合，我们开发了一种适用于定量、功能和分子分析单个 MBC 的方案，这些 MBC 针对同一管中多达两种不同流感株的 HA 具有特异性。这种方法将能够鉴定感染或接种疫苗后出现在血液中的浆母细胞和新型 MBC 的幼稚和 MBC 前体，从而阐明先前存在的 MBC 在针对新型流感病毒的抗体反应中的实际贡献。最后，该方案可以允许应用高通量深度测序来分析来自大量疫苗接种者和感染受试者的纵向样本中 HA⁺ B 细胞库的变化，最终目的是了解体内 B 细胞动力学如何推动广泛交叉保护抗体反应的演变。
