Characterizing adjuvants' effects at murine immunoglobulin repertoire level.

Generating large-scale, high-fidelity sequencing data is challenging and, furthermore, not much has been done to characterize adjuvants' effects at the repertoire level. Thus, we introduced an IgSeq pipeline that standardized library prep protocols and data analysis functions for accurate repertoire profiling. We then studied systemically effects of CpG and Alum on the Ig heavy chain repertoire using the ovalbumin (OVA) murine model. Ig repertoires of different tissues (spleen and bone marrow) and isotypes (IgG and IgM) were examined and compared in IGHV mutation, gene usage, CDR3 length, clonal diversity, and clonal selection. We found Ig repertoires of different compartments exhibited distinguishable profiles at the non-immunized steady state, and distinctions became more pronounced upon adjuvanted immunizations. Notably, Alum and CpG effects exhibited different tissue- and isotype-preferences. The former led to increased diversity of abundant clones in bone marrow, and the latter promoted the selection of IgG clones in both tissues.

Structure and Diversity of the Rhesus Macaque Immunoglobulin Loci through Multiple De Novo Genome Assemblies.

The rhesus macaque is a critically important animal model in biomedical research, most recently playing a key role in the development of vaccines against human immunodeficiency virus-1. Nevertheless, the immunoglobulin (Ig) loci of macaques are as yet incompletely determined and our understanding of differences between human and macaque humoral immunity remains deficient. We completed a high-coverage, high-quality whole genome sequencing and assembly project with a single rhesus macaque of Indian origin, and partial genome assemblies using genomic molecular targeting of the Ig loci in nine other rhesus macaques of Indian origin. These data indicate that the macaque Ig loci are substantially more diverse than those in humans, including greater sequence diversity and copy-number variation between individuals. It appears likely that such copy-number variation even occurs between allelic loci within individuals. Different Ig gene families in the macaque show distinct relationships to the corresponding human gene families and appear to evolve under different mechanisms. These results raise intriguing questions about the evolution of antigen receptors in primates but also have important practical implications for the design and interpretation of biomedical studies.

HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy.

Fluorescence in situ hybridization and optical mapping to correct scaffold arrangement in the tomato genome.

The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.