Fisher, C. R. | bioRxiv Science

bioRxiv2018

EnTAP: Bringing Faster and Smarter Functional Annotation to Non-Model Eukaryotic Transcriptomes

EnTAP (Eukaryotic Non-Model Transcriptome Annotation Pipeline) was designed to improve the accuracy, speed, and flexibility of functional gene annotation for de novo assembled transcriptomes in non-model eukaryotes. This software package addresses the fragmentation and related assembly issues that result in inflated transcript estimates and poor annotation rates, while focusing primarily on protein-coding transcripts. Following filters applied through assessment of true expression and frame selection, open-source tools are leveraged to functionally annotate the translated proteins. Downstream features include fast similarity search across three repositories, protein domain assignment, orthologous gene family assessment, and Gene Ontology term assignment. The final annotation integrates across multiple databases and selects an optimal assignment from a combination of weighted metrics describing similarity search score, taxonomic relationship, and informativeness. Researchers have the option to include additional filters to identify and remove contaminants, identify associated pathways, and prepare the transcripts for enrichment analysis. This fully featured pipeline is easy to install, configure, and runs significantly faster than comparable annotation packages. EnTAP is optimized to generate extensive functional information for the gene space of organisms with limited or poorly characterized genomic resources.

bioinformatics↗

bioRxiv2017

Multivalency drives the neutralizing activity of antibodies against the Plasmodium falciparum circumsporozoite protein

The repeat region of the Plasmodium falciparum circumsporozoite protein (CSP) is a major vaccine antigen because it can be targeted by parasite neutralizing antibodies; however, little is known about this interaction. We used isothermal titration calorimetry, X-ray crystallography and mutagenesis-validated modeling to analyze the binding of a murine neutralizing antibody to Plasmodium falciparum CSP. Strikingly, we found that the repeat region of CSP is bound by multiple antibodies. This repeating pattern allows multiple weak interactions of single FAB domains to accumulate and yield a complex with a dissociation constant in the low nM range. Because the CSP protein can potentially cross-link multiple B cell receptors (BCRs) we hypothesized that the B cell response might be T cell independent. However, while there was a modest response in mice deficient in T cell help, the bulk of the response was T cell dependent. By sequencing the BCRs of CSP-repeat specific B cells in inbred mice we found that these cells underwent somatic hypermutation and affinity maturation indicative of a T-dependent response. Last, we found that the BCR repertoire of responding B cells was limited suggesting that the structural simplicity of the repeat may limit the breadth of the immune response.\n\nAuthor SummaryVaccines aim to protect by inducing the immune system to make molecules called antibodies that can recognize molecules on the surface of invading pathogens. In the case of malaria, our most advanced vaccine candidates aim to promote the production of antibodies that recognize the circumsporozoite protein (CSP) molecule on the surface of the invasive parasite stage called the sporozoite. In this report we use X-ray crystallography to determine the structure of CSP-binding antibodies at the atomic level. We use other techniques such as isothermal titration calorimetry and structural modeling to examine how this antibody interacts with the CSP molecule. Strikingly, we found that each CSP molecule could bind 6 antibodies. This finding has implications for the immune response and may explain why high titers of antibody are needed for protection. Moreover, because the structure of the CSP repeat is quite simple we determined that the number of different kinds of antibodies that could bind this molecule are quite small. However a high avidity interaction between those antibodies and CSP can result from a process called affinity maturation that allows the body to learn how to make improved antibodies specific for pathogen molecules. These data show that while it is challenging for the immune system to recognize and neutralize CSP, it should be possible to generate viable vaccines targeting this molecule.

immunology↗