mRNA prime–boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models

Germline-targeting (GT) protein immunogens to induce VRC01-class broadly neutralizing antibodies (bnAbs) to the CD4 binding site (CD4bs) of the HIV Envelope have shown promise in clinical trials. Here, we preclinically validated the mRNA-LNP delivery of one such immunogen, eOD-GT8, as a soluble self-assembling 60mer nanoparticle in humanized mouse models. In a model with three humanized B cell lineages bearing distinct VRC01-precursor B cell receptors (BCRs) with similar affinities for eOD-GT8, all lineages could be simultaneously primed and undergo diversification and affinity maturation with no sign of exclusionary competition. Boosts drove precursor B cell participation in germinal centers, the accumulation of somatic hypermutations, including in key VRC01-class positions, and affinity maturation to boost and native-like antigens in two of the three precursor lineages. We have preclinically validated a prime-boost regimen of soluble self-assembling nanoparticles delivered by mRNA-LNP, demonstrating that multiple lineages can be primed, boosted, and diversify along the bnAb pathway.

Methods
BCR Sequences from mice after prime-boost were isolated by single cell sequencing, see Materials and Methods/ BCR sequencing section. The unpaired sequences of  heavy and light chains are shown in excel sheets.

靶向生殖细胞系（Germline-targeting, GT）的蛋白免疫原，用于诱导针对HIV包膜CD4结合位点（CD4 binding site, CD4bs）的VRC01类广谱中和抗体（broadly neutralizing antibodies, bnAbs），已在临床试验中展现出应用前景。本研究在人源化小鼠模型中，对以可溶性自组装60聚体纳米颗粒形式存在的该类免疫原eOD-GT8的mRNA-LNP递送策略进行了临床前验证。在包含三类携带不同VRC01前体B细胞受体（B cell receptor, BCR）、且对eOD-GT8具有相近亲和力的人源化B细胞谱系模型中，所有谱系均可同时被致敏，并发生谱系分化与亲和力成熟，未出现排他性竞争的迹象。加强免疫可促使前体B细胞进入生发中心，积累体细胞超突变（包括VRC01类关键位点的突变），并使三类前体谱系中的两类实现针对加强免疫抗原与天然类似抗原的亲和力成熟。本研究完成了对mRNA-LNP递送可溶性自组装纳米颗粒的初免-加强免疫方案的临床前验证，证实多个谱系可被致敏、接受加强免疫，并沿广谱中和抗体通路发生谱系分化。

实验方法
初免-加强免疫后小鼠体内的B细胞受体序列通过单细胞测序技术获取，详见实验方法/B细胞受体测序章节。重链与轻链的未配对序列已整理至Excel表格中。