Mucosal transmission of the human immunodeficiency virus (HIV) results in a bottleneck in viral genetic diversity. Gnanakaran and colleagues used a computational strategy to identify signature amino acids at particular positions in Envelope that were associated either with transmitted sequences sampled very early in infection, or sequences sampled during chronic infection. Among the strongest signatures observed was an enrichment for the stable presence of histidine at position 12 at transmission and in early infection, and a recurrent loss of histidine at position 12 in chronic infection. This amino acid lies within the leader peptide of Envelope, a region of the protein that has been shown to influence envelope glycoprotein expression and virion infectivity. We show a strong association between a positively charged amino acid like histidine at position 12 in transmitted/founder viruses with more efficient trafficking of the nascent envelope polypeptide to the endoplasmic reticulum and higher steady-state glycoprotein expression compared to viruses that have a non-basic position 12 residue, a substitution that was enriched among viruses sampled from chronically infected individuals. When expressed in the context of other viral proteins, transmitted envelopes with a basic amino acid position 12 were incorporated at higher density into the virus and exhibited higher infectious titers than did non-signature envelopes. These results support the potential utility of using a computational approach to examine large viral sequence data sets for functional signatures and indicate the importance of Envelope expression levels for efficient HIV transmission.

BackgroundNext-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.ResultsWe isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations. Next, we prepared three technical replicates of antibody libraries by RT-PCR from each diversity scenario, which were sequenced using the Illumina MiSeq platform resulting in >106 250 bp paired-end reads per replicate. We then assessed the robustness of antibody repertoire data based on clonal identification defined by amino acid sequence of either full-length VDJ region or the complementarity determining region 3 (CDR3). Leveraging modeling approaches adapted from mathematical ecology, we found that in either diversity scenario both CDR3 and VDJ detection nears completeness indicating deep coverage of ASC repertoires. Additionally, we defined reliability thresholds for accurate quantification and ranking of CDR3s and VDJs. Importantly, we show that both factors(i) replicate sequencing and (ii) sequencing depth–are crucial for robust CDR3 and VDJ detection and ranking.ConclusionsIn summary, we established widely applicable experimental and computational guidelines for robust antibody NGS and analysis, which will help advance systems immunology studies related to the quantitative profiling of antibody responses following infection and vaccination.Electronic supplementary materialThe online version of this article (doi:10.1186/s12865-014-0040-5) contains supplementary material, which is available to authorized users.

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences. As of yet, the impact of the method of adapter addition on experimental library preparation and the resulting antibody repertoire HTS datasets has not been thoroughly investigated. Therefore, we compared three standard library preparation methods by performing Illumina HTS on antibody variable heavy genes from murine antibody-secreting cells. Clonal overlap and rank statistics demonstrated that the investigated methods produced equivalent HTS datasets. PCR-based methods were experimentally superior to ligation with respect to speed, efficiency, and practicality. Finally, using a two-step PCR based method we established a protocol for antibody repertoire library generation, beginning from inputs as low as 1 ng of total RNA. In summary, this study represents a major advance towards a standardized experimental framework for antibody HTS, thus opening up the potential for systems-based, cross-experiment meta-analyses of antibody repertoires.

In vitro antibody display and screening technologies geared toward the discovery and engineering of clinically applicable antibodies have evolved from screening artificial antibody formats, powered by microbial display technologies, to screening of natural, full-IgG molecules expressed in mammalian cells to readily yield lead antibodies with favorable properties in production and clinical applications. Here, we report the development and characterization of a novel, next-generation mammalian cell-based antibody display and screening platform called Transpo-mAb Display, offering straightforward and efficient generation of cellular libraries by using non-viral transposition technology to obtain stable antibody expression. Because Transpo-mAb Display uses DNA-transposable vectors with substantial cargo capacity, genomic antibody heavy chain expression constructs can be utilized that undergo the natural switch from membrane bound to secreted antibody expression in B cells by way of alternative splicing of Ig-heavy chain transcripts from the same genomic expression cassette. We demonstrate that stably transposed cells co-express transmembrane and secreted antibodies at levels comparable to those provided by dedicated constructs for secreted and membrane-associated IgGs. This unique feature expedites the screening and antibody characterization process by obviating the need for intermediate sequencing and re-cloning of individual antibody clones into separate expression vectors for functional screening purposes. In a series of proof-of-concept experiments, we demonstrate the seamless integration of antibody discovery with functional screening for various antibody properties, including binding affinity and suitability for preparation of antibody-drug conjugates.

While the contribution of CD8+ cytotoxic T lymphocytes to early containment of HIV-1 spread is well established, a role for NK cells in controlling HIV-1 replication during primary infection has been uncertain. The highly polymorphic family of KIR molecules expressed on NK cells can inhibit or activate these effector cells and might therefore modulate their activity against HIV-1-infected cells. In the present study, we investigated copy number variation in KIR3DH loci encoding the only activating KIR receptor family in rhesus monkeys and its effect on simian immunodeficiency virus (SIV) replication during primary infection in rhesus monkeys. We observed an association between copy numbers of KIR3DH genes and control of SIV replication in Mamu-A*01– rhesus monkeys that express restrictive TRIM5 alleles. These findings provide further evidence for an association between NK cells and the early containment of SIV replication, and underscore the potential importance of activating KIRs in stimulating NK cell responses to control SIV spread.

Here, we demonstrate that KIR2DL4 copy number variation (CNV) is associated with CD4 + T-cell decline and functionality of cytokine-producing NK cells during primary simian immunodeficiency virus (SIV) infection in Mamu-A * 01 − Indian-origin rhesus macaques, with higher KIR2DL4 copy numbers being associated with a better preservation of CD4 + T cells and an increased gamma interferon (IFN-γ) production from stimulated cytokine-producing NK cell subsets during acute SIVmac251 infection. These findings underscore the crucial role of activating killer-cell immunoglobulin-like receptors (KIRs) in NK cell-mediated SIV responses during early SIV infection.

Receptor tyrosine kinase-like orphan receptor 2 (ROR2) has been identified as a highly relevant tumor-associated antigen in a variety of cancer indications of high unmet medical need, including renal cell carcinoma and osteosarcoma, making it an attractive target for targeted cancer therapy. Here, we describe the de novo discovery of fully human ROR2-specific antibodies and potent antibody drug conjugates (ADCs) derived thereof by combining antibody discovery from immune libraries of human immunoglobulin transgenic animals using the Transpo-mAb mammalian cell-based IgG display platform with functional screening for internalizing antibodies using a secondary ADC assay. The discovery strategy entailed immunization of transgenic mice with the cancer antigen ROR2, harboring transgenic IgH and IgL chain gene loci with limited number of fully human V, D, and J gene segments. This was followed by recovering antibody repertoires from the immunized animals, expressing and screening them as full-length human IgG libraries by transposon-mediated display in progenitor B lymphocytes (“Transpo-mAb Display”) for ROR2 binding. Individual cellular “Transpo-mAb” clones isolated by single cell sorting and capable of expressing membrane-bound as well as secreted human IgG were directly screened during antibody discovery, not only for high affinity binding to human ROR2, but also functionally as ADCs using a cytotoxicity assay with a secondary anti-human IgG-toxin-conjugate. Using this strategy, we identified and validated 12 fully human, monoclonal anti-human ROR2 antibodies with nanomolar affinities that are highly potent as ADCs and could be promising candidates for the therapy of human cancer. The screening for functional and internalizing antibodies during the early phase of antibody discovery demonstrates the utility of the mammalian cell-based Transpo-mAb Display platform to select for functional binders and as a powerful tool to improve the efficiency for the development of therapeutically relevant ADCs.

Here we show that the number of activating killer cell immunoglobulin-like receptor (KIR) copies in rhesus monkeys is associated with the extent of release of cytotoxic granules by cytolytic NK cells during primary simian immunodeficiency virus SIVmac251 infection. These findings suggest that NK cells expressing high levels of activating KIRs efficiently kill SIVmac251-infected cells, and this efficient killing contributes to the NK cell-mediated control of replication of this virus during early infection. While it is well established that NK cells play a central role in the early control of a number of viral infections (11,18,22,23), their role in containing HIV-1 replication is only now being clarified. Studies have shown that activating killer cell immunoglobulin-like receptors (KIRs) that are expressed on NK cells are involved in controlling HIV-1 replication and slowing HIV-1 disease progression (1,2,15,17). While there is reason to suppose that NK cells affect HIV-1 replication early following infection, these cells are difficult to study during primary infection because it is difficult to obtain early blood samples from infected individuals. The simian immunodeficiency virus (SIV)-infected rhesus monkey provides an important nonhuman primate animal model for studying NK cell biology during a primary AIDS virus infection. We have recently demonstrated an association between specific NK cell subpopulations and the early containment of SIV replication in rhesus monkeys and a contribution of activating KIRs in stimulating NK cells to control the spread of SIV (8). In that study, we evaluated copy number variation (CNV) of KIR3DH, which represents an activating KIR receptor family in rhesus monkeys (4, 9), and showed that KIR3DH copy numbers were negatively associated with SIV replication at the time of the early peak of viral replication in Mamu-A*01 Ϫ rhesus monkeys that express restrictive TRIM5 alleles. This observation implicated NK cells that express activating KIRs in controlling viral replication during early SIV infection. However, the mechanism underlying this phenomenon remains unclear.The present study was initiated to determine how activating KIRs might affect SIV replication during primary infection. One important function of NK cells is to kill virus-infected target cells, and the present study assessed whether the cytolytic activity of NK cells is affected by KIR3DH CNV. In this study, the cytotoxicity of peripheral blood NK cells of Mamu-A*01 Ϫ rhesus monkeys was evaluated by measuring intracellular granzyme B and perforin levels and assessing CD107a surface expression on NK cells following in vitro stimulation with K562 cells, a cell line that does not express major histocompatibility complex (MHC) class I molecules (3,13,14). Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-anticoagulated whole blood by Ficoll-Paque (GE Healthcare, Piscataway, NJ) gradient separation and either stained immediately or cryopreserved in liquid nitrogen. After thawing, cryopreserved cells were ...