DOI: 10.5061/dryad.rn8pk0pk5
title: mRNA prime–boost evolves precursors toward VRC01-like broadly neutralizing antibodies in preclinical humanized mouse models

description: 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.
description: 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.
description: # mRNA prime–boost evolves precursors toward VRC01-like broadly
 neutralizing antibodies in preclinical humanized mouse models
 [https://doi.org/10.5061/dryad.rn8pk0pk5](https://doi.org/10.5061/dryad.rn8pk0pk5) BCR Sequences from germinal center (GC) in CLK adoptively transferred mice after prime-boost. ## Description of the data and file structure BCR sequencing data for mutation analysis of heavy chains (HCs) and light chains (LCs) are from co-transferred mice bearing CLK19,CLK09 and CLK21 B cells, those mice were primed with eOD-GT8-60 mer mRNA-LNP and boosted with either g5- 60mer or g28-60 mer or g28v2-60mer mRNA-LNP. GC B single cells from mice immunized at the different time points were sorted in 96-well plates by antigen-specific immunogen probes . mRNA of HCs and LCs were amplified from single cell for sanger sequencing. BCR sequences were processed using the IMGT/HighV-QUEST. Data files include: nucleotide sequences of heavy and light chain from mice at 14 days post-prime with eOD-GT8-60 mer mRNA-LNP nucleotide sequences of heavy and light chain from mice at 36 days post-prime with eOD-GT8-60 mer mRNA-LNP nucleotide sequences of heavy and light chain from mice at 36 days post-boost with g5-60 mer mRNA-LNP nucleotide sequences of heavy and light chain from mice at 36 days post-boost with g28-60 mer mRNA-LNP nucleotide sequences of heavy and light chain from mice at 8 days post-boost with g28v2-60 mer mRNA-LNP nucleotide sequences of heavy and light chain from mice at 16 days post-boost with g28v2-60 mer mRNA-LNP nucleotide sequences of heavy and light chain from mice at 28 days post-boost with g28v2-60 mer mRNA-LNP Details of data sources and file information are shown as follows: | run\_date | prime | Boost | Time\_point | file\_tag | | :-------- | :----------- | :--------- | :------------- | :------------------------------ | | 20-07-08 | eOD-GT8-mRNA | none | D14-post-prime | 200708\_GT8\_D14\_post\_prime | | 20-07-29 | eOD-GT8-mRNA | none | D36-post-prime | 200729\_GT8\_D36\_post\_prime | | 20-09-09 | eOD-GT8-mRNA | g5-mRNA | D36-post-boost | 200909\_g5\_D36\_post\_boost | | 20-09-09 | eOD-GT8-mRNA | g28-mRNA | D36-post-boost | 200909\_g28\_D36\_post\_boost | | 21-02-24 | eOD-GT8-mRNA | g28v2-mRNA | D8-post-boost | 210224\_g28v2\_D8\_post\_boost | | 21-03-04 | eOD-GT8-mRNA | g28v2-mRNA | D16-post-boost | 210304\_g28v2\_D16\_post\_boost | | 21-03-16 | eOD-GT8-mRNA | g28v2-mRNA | D28-post-boost | 210316\_g28v2\_D28\_post\_boost | Each file contains two excel sheets including both heavy chain(HC) and light chain(LC) sequences.HC and LC sequences are unpaired. ## Code/Software As described in the "BCR sequencing" section of Methods and Materials: Briefly, sequences from immunized mice underwent quality control, mutations were aligned to the reference CLK19, CLK09 and CLK21 sequences and analyzed using Geneious software, IMGT/V-QUEST was used for Human Ig gene analysis.