HLA class II-Restricted CD8+ T cells in HIV-1 Virus Controllers.

A paradigm shifting study demonstrated that induction of MHC class E and II-restricted CD8+ T cells was associated with the clearance of SIV infection in rhesus macaques. Another recent study highlighted the presence of HIV-1-specific class II-restricted CD8+ T cells in HIV-1 patients who naturally control infection (virus controllers; VCs). However, questions regarding class II-restricted CD8+ T cells ontogeny, distribution across different HIV-1 disease states and their role in viral control remain unclear. In this study, we investigated the distribution and anti-viral properties of HLA-DRB1*0701 and DQB1*0501 class II-restricted CD8+ T cells in different HIV-1 patient cohorts; and whether class II-restricted CD8+ T cells represent a unique T cell subset. We show that memory class II-restricted CD8+ T cell responses were more often detectable in VCs than in chronically infected patients, but not in healthy seronegative donors. We also demonstrate that VC CD8+ T cells inhibit virus replication in both a class I- and class II-dependent manner, and that in two VC patients the class II-restricted CD8+ T cells with an anti-viral gene signature expressed both CD4+ and CD8+ T cell lineage-specific genes. These data demonstrated that anti-viral memory class II-restricted CD8+ T cells with hybrid CD4+ and CD8+ features are present during natural HIV-1 infection.

Detection of a Low Level and Heterogeneous B Cell Immune Response in Peripheral Blood of Acute Borreliosis Patients With High Throughput Sequencing.

The molecular diagnosis of acute Borreliosis is complicated and better strategies to improve the diagnostic processes are warranted. High Throughput Sequencing (HTS) of human B cell repertoires after e.g., Dengue virus infection or influenza vaccination revealed antigen-associated "CDR3 signatures" which may have the potential to support diagnosis in infectious diseases. The human B cell immune response to Borrelia burgdorferi sensu lato-the causative agent of Borreliosis-has mainly been studied at the antibody level, while less attention has been given to the cellular part of the humoral immune response. There are indications that Borrelia actively influence the B cell immune response and that it is therefore not directly comparable to responses induced by other infections. The main goal of this study was to identify B cell features that could be used to support diagnosis of Borreliosis. Therefore, we characterized the B cell immune response in these patients by combining multicolor flow cytometry, single Borrelia-reactive B cell receptor (BCR) sequencing, and B cell repertoire deep sequencing. Our phenotyping experiments showed, that there is no significant difference between B cell subpopulations of acute Borreliosis patients and controls. BCR sequences from individual epitope-reactive B cells had little in common between each other. HTS showed, however, a higher complementarity determining region 3 (CDR3) amino acid (aa) sequence overlap between samples from different timepoints in patients as compared to controls. This indicates, that HTS is sensitive enough to detect ongoing B cell immune responses in these patients. Although each individual's repertoire was dominated by rather unique clones, clustering of bulk BCR repertoire sequences revealed a higher overlap of IgG BCR repertoire sequences between acute patients than controls. Even if we have identified a few Borrelia-associated CDR3aa sequences, they seem to be rather unique for each patient and therefore not suitable as biomarkers.

High-throughput sequencing of murine immunoglobulin heavy chain repertoires using single side unique molecular identifiers on an Ion Torrent PGM.

With the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT's underlying algorithms efficiently detect 98% of the introduced indels. Undetected indels are either located at the end of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. The complementary determining regions 3 (CDR3s) are returned correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers result in highly accurate HTS data if combined with the presented processing strategy. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. Finally, we show that the protocol can readily be used to generate homogenous datasets for bulk sequencing of murine bone marrow samples. Taken together, this approach will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

Functional Relevance of Improbable Antibody Mutations for HIV Broadly Neutralizing Antibody Development.

HIV-1 broadly neutralizing antibodies (bnAbs) require high levels of activation-induced cytidine deaminase (AID)-catalyzed somatic mutations for optimal neutralization potency. Probable mutations occur at sites of frequent AID activity, while improbable mutations occur where AID activity is infrequent. One bottleneck for induction of bnAbs is the evolution of viral envelopes (Envs) that can select bnAb B cell receptors (BCR) with improbable mutations. Here we define the probability of bnAb mutations and demonstrate the functional significance of key improbable mutations in three bnAb B cell lineages. We show that bnAbs are enriched for improbable mutations, which implies that their elicitation will be critical for successful vaccine induction of potent bnAb B cell lineages. We discuss a mutation-guided vaccine strategy for identification of Envs that can select B cells with BCRs that have key improbable mutations required for bnAb development.

Functionally Convergent B Cell Receptor Sequences in Transgenic Rats Expressing a Human B Cell Repertoire in Response to Tetanus Toxoid and Measles Antigens.

The identification and tracking of antigen-specific immunoglobulin (Ig) sequences within total Ig repertoires is central to high-throughput sequencing (HTS) studies of infections or vaccinations. In this context, public Ig sequences shared by different individuals exposed to the same antigen could be valuable markers for tracing back infections, measuring vaccine immunogenicity, and perhaps ultimately allow the reconstruction of the immunological history of an individual. Here, we immunized groups of transgenic rats expressing human Ig against tetanus toxoid (TT), Modified Vaccinia virus Ankara (MVA), measles virus hemagglutinin and fusion proteins expressed on MVA, and the environmental carcinogen benzo[a]pyrene, coupled to TT. We showed that these antigens impose a selective pressure causing the Ig heavy chain (IgH) repertoires of the rats to converge toward the expression of antibodies with highly similar IgH CDR3 amino acid sequences. We present a computational approach, similar to differential gene expression analysis, that selects for clusters of CDR3s with 80% similarity, significantly overrepresented within the different groups of immunized rats. These IgH clusters represent antigen-induced IgH signatures exhibiting stereotypic amino acid patterns including previously described TT- and measles-specific IgH sequences. Our data suggest that with the presented methodology, transgenic Ig rats can be utilized as a model to identify antigen-induced, human IgH signatures to a variety of different antigens.

High-resolution analysis of the B cell repertoire before and after polyethylene glycol fusion reveals preferential fusion of rare antigen-specific B cells.

OBJECTIVE: The hybridoma technology is one of the most important advances in clinical immunology. Little is known about the differences between the antibodies produced during the in vivo immune response and those recovered in hybridoma libraries. Here, we investigate a potential fusion bias inherent to the hybridoma production process. METHODS: Transgenic rats carrying human Ig heavy and light chain loci were immunized with measles virus (MV) to generate human mAbs. Usin g high-throughput sequencing of IgH mRNA, we compared the IgH repertoire of lymph nodes and the derived hybridoma library using the sequences of the MV-specific hybridoma clones as a reference set with known specificity. RESULTS: We observed that large clonotypes from the lymph nodes were not represented in the hybridoma library, but low-frequency B cell populations became highly enriched and most hybridoma clones were derived from these. Our data also showed that identical CDR3s evolved from diverse VDJ recombinations, indicating convergence of different B cells subpopulations towards expression of antibodies with similar paratopes. CONCLUSION: The efficient generation of mAbs results from a fusion process highly selective for rare antigen-specific B cells rather than in vivo expanded populations. Antibodies of particular interest may therefore be missed during classical hybridoma production.

Whole genome single-nucleotide variation profile-based phylogenetic tree building methods for analysis of viral, bacterial and human genomes.

UNLABELLED: Next-generation sequencing data can be mapped to a reference genome to identify single-nucleotide polymorphisms/variations (SNPs/SNVs; called SNPs hereafter). In theory, SNPs can be compared across several samples and the differences can be used to create phylogenetic trees depicting relatedness among the samples. However, in practice this is difficult because currently there is no stand-alone tool that takes SNP data directly as input and produces phylogenetic trees. In response to this need, PhyloSNP application was created with two analysis methods 1) a quantitative method that creates the presence/absence matrix which can be directly used to generate phylogenetic trees or creates a tree from a shrunk genome alignment (includes additional bases surrounding the SNP position) and 2) a qualitative method that clusters samples based on the frequency of different bases found at a particular position. The algorithms were used to generate trees from Poliovirus, Burkholderia and human cancer genomics NGS datasets. AVAILABILITY: PhyloSNP is freely available for download at http://hive.biochemistry.gwu.edu/dna.cgi?cmd=phylosnp.

Non-synonymous variations in cancer and their effects on the human proteome: workflow for NGS data biocuration and proteome-wide analysis of TCGA data.

BACKGROUND: Next-generation sequencing (NGS) technologies have resulted in petabytes of scattered data, decentralized in archives, databases and sometimes in isolated hard-disks which are inaccessible for browsing and analysis. It is expected that curated secondary databases will help organize some of this Big Data thereby allowing users better navigate, search and compute on it. RESULTS: To address the above challenge, we have implemented a NGS biocuration workflow and are analyzing short read sequences and associated metadata from cancer patients to better understand the human variome. Curation of variation and other related information from control (normal tissue) and case (tumor) samples will provide comprehensive background information that can be used in genomic medicine research and application studies. Our approach includes a CloudBioLinux Virtual Machine which is used upstream of an integrated High-performance Integrated Virtual Environment (HIVE) that encapsulates Curated Short Read archive (CSR) and a proteome-wide variation effect analysis tool (SNVDis). As a proof-of-concept, we have curated and analyzed control and case breast cancer datasets from the NCI cancer genomics program - The Cancer Genome Atlas (TCGA). Our efforts include reviewing and recording in CSR available clinical information on patients, mapping of the reads to the reference followed by identification of non-synonymous Single Nucleotide Variations (nsSNVs) and integrating the data with tools that allow analysis of effect nsSNVs on the human proteome. Furthermore, we have also developed a novel phylogenetic analysis algorithm that uses SNV positions and can be used to classify the patient population. The workflow described here lays the foundation for analysis of short read sequence data to identify rare and novel SNVs that are not present in dbSNP and therefore provides a more comprehensive understanding of the human variome. Variation results for single genes as well as the entire study are available from the CSR website (http://hive.biochemistry.gwu.edu/dna.cgi?cmd=csr). CONCLUSIONS: Availability of thousands of sequenced samples from patients provides a rich repository of sequence information that can be utilized to identify individual level SNVs and their effect on the human proteome beyond what the dbSNP database provides.