A successful HIV vaccine eliciting broadly neutralizing antibodies (bnAbs) must overcome the hurdle of being able to activate naive precursor B cells encoding features within their germline B cell receptors (BCR) that allow recognition of broadly neutralizing epitopes. Knowledge of whether bnAb precursor B cells are circulating at sufficient frequencies within individuals in communities heavily impacted by HIV may be important. Using a germline-targeting eOD-GT8 immunogen and high-throughput droplet-based single-cell BCR sequencing, we demonstrate that large numbers of paired BCR sequences from multiple donors can be efficiently screened to elucidate precursor frequencies of rare, naive VRC01-class B cells. Further, we analyzed IGHV1-2 allelic usage among three different cohorts; we find that IGHV1-2 alleles traditionally thought to be incompatible with VRC01-class responses are relatively common in various human populations and that germline variation within IGHV1-2 associates with gene usage frequencies in the naive BCR repertoire.

The contribution of heritable factors to antibody function and diversity is not fully understood, but has profound implications for delineating variation in the antibody response observed at the population-level. We performed matched long-read-based characterization of the immunoglobulin heavy chain (IGH) locus and expressed antibody repertoire profiling at population-scale to examine, for the first time, the impact of IGH genomic variation on the antibody repertoire. We characterized extensive IGH polymorphism, including novel structural variants (SVs), small insertion/deletions (indels), single nucleotide variants (SNVs), and IG genes and alleles. Countering models that antibody repertoire diversity is driven largely by stochastic processes, we demonstrate that IGH genetic factors make significant contributions to gene usage in both the naive and antigen-experienced repertoire. Specifically, the usage of 73% of IGH genes was associated with common polymorphisms, including those capable of explaining >70% of variance in gene usage. These variants were enriched in transcription factor binding sites and other functional elements associated with V(D)J recombination, and overlapped polymorphisms from genome-wide association studies. Furthermore, we found evidence for the coordinated regulation of IGH genes across the repertoire, demonstrating complex interactions between IGH variants and gene usage. These results refine our understanding of variation observed in the antibody repertoire, and will advance the study of antibody function in disease.

The adaptive immune receptor repertoire (AIRR) contains information on an individuals' immune past, present and potential in the form of the evolving sequences that encode the B cell receptor (BCR) repertoire. AIRR sequencing (AIRR-seq) studies rely on databases of known BCR germline variable (V), diversity (D), and joining (J) genes to detect somatic mutations in AIRR-seq data via comparison to the best-aligning database alleles. However, it has been shown that these databases are far from complete, leading to systematic misidentification of mutated positions in subsets of sample sequences. We previously presented TIgGER, a computational method to identify subject-specific V gene genotypes, including the presence of novel V gene alleles, directly from AIRR-seq data. However, the original algorithm was unable to detect alleles that differed by more than 5 single nucleotide polymorphisms (SNPs) from a database allele. Here we present and apply an improved version of the TIgGER algorithm which can detect alleles that differ by any number of SNPs from the nearest database allele, and can construct subject-specific genotypes with minimal prior information. TIgGER predictions are validated both computationally (using a leave-one-out strategy) and experimentally (using genomic sequencing), resulting in the addition of three new immunoglobulin heavy chain V (IGHV) gene alleles to the IMGT repertoire. Finally, we develop a Bayesian strategy to provide a confidence estimate associated with genotype calls. All together, these methods allow for much higher accuracy in germline allele assignment, an essential step in AIRR-seq studies.

17The adaptive immune receptor repertoire (AIRR) contains information on an individuals' 18 immune past, present and potential in the form of the evolving sequences that encode the 19 B cell receptor (BCR) repertoire. AIRR sequencing (AIRR-seq) studies rely on databases 20 of known BCR germline variable (V), diversity (D) and joining (J) genes to detect somatic 21 mutations in AIRR-seq data via comparison to the best-aligning database alleles. 22As depicted in Figure 3, TIgGER was run iteratively to detect the set of alleles carried by three subjects. In

To better understand the subspecies origin of antibody genes in classical inbred mouse strains, the IGH gene loci of four wild-derived mouse strains were explored by analysis of VDJ gene rearrangements. A total of 341 unique IGHV gene sequences were inferred in the wild-derived strains, including 247 sequences that have not previously been reported. The genes of the Non-Obese Diabetic (NOD) strain were also documented, and all but one of the 84 inferred NOD IGHV genes have previously been observed in C57BL/6 mice. This is surprising because the Swiss mouse-derived NOD strain and the C57BL/6 strain have no known shared ancestry. The relationships between the genes of the wild-derived inbred strains and of the C57BL/6, NOD and BALB/c classical inbred strain were then explored. The IGH loci of the C57BL/6 and the MSM/MsJ strains share many sequences, but analysis showed that few sequences are shared with wild-derived strains representing the three major subspecies of the house mouse. There were also few IGHV sequences that were shared by the BALB/c strain and any of the four wild-derived strains. The origins of IGHV genes in the C57BL/6, MSM/MsJ and BALB/c strains therefore remain unclear. These unexpected similarities and differences highlight our lack of understanding of the antibody gene loci of the laboratory mouse, with implications for the interpretation of strain-specific differences in models of antibody-mediated diseases, and of Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) data. These results also suggest that a position-based immunoglobulin gene nomenclature may be unworkable in the mouse.

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SUMMARY Gut microbiota composition is associated with human and rodent Plasmodium infections, yet the mechanism by which gut microbiota affects the severity of malaria remains unknown. Humoral immunity is critical in mediating the clearance of Plasmodium blood stage infections, prompting the hypothesis that mice with gut microbiota-dependent decreases in parasite burden exhibit better germinal center (GC) responses. In support of this hypothesis, mice with a low parasite burden exhibit increases in GC B cell numbers and parasite-specific antibody titers, as well as better maintenance of GC structures and a more targeted, qualitatively different antibody response. This enhanced humoral immunity affects memory, as mice with a low parasite burden exhibit robust protection against challenge with a heterologous, lethal Plasmodium species. These results demonstrate that gut microbiota composition influences the biology of spleen GCs as well as the titer and repertoire of parasite-specific antibodies, identifying potential approaches to develop optimal treatments for malaria.

The genomes of classical inbred mouse strains include genes derived from all three major subspecies of the house mouse, Mus musculus. We recently posited that genetic diversity in the immunoglobulin heavy chain (IGH) gene loci of C57BL/6 and BALB/c mice reflects differences in subspecies origin. To investigate this hypothesis, we conducted high‐throughput sequencing of IGH gene rearrangements to document IGH variable (IGHV), joining (IGHJ) and diversity (IGHD) genes in four inbred wild‐derived mouse strains (CAST/EiJ, LEWES/EiJ, MSM/MsJ and PWD/PhJ) and a single disease model strain (NOD/ShiLtJ), collectively representing genetic backgrounds of several major mouse subspecies. A total of 341 germline IGHV sequences were inferred in the wild‐derived strains, including 247 not curated in the international ImMunoGeneTics information system. By contrast, 83/84 inferred NOD IGHV genes had previously been observed in C57BL/6 mice. Variability among the strains examined was observed for only a single IGHJ gene, involving a description of a novel allele. By contrast, unexpected variation was found in the IGHD gene loci, with four previously unreported IGHD gene sequences being documented. Very few IGHV sequences of C57BL/6 and BALB/c mice were shared with strains representing major subspecies, suggesting that their IGH loci may be complex mosaics of genes of disparate origins. This suggests a similar level of diversity is likely present in the IGH loci of other classical inbred strains. This must now be documented if we are to properly understand interstrain variation in models of antibody‐mediated disease.