Identifying the structures that contribute to monoclonal antibody (mAb) binding sites (epitopes) within native G protein-coupled receptors (GPCRs) can be useful for developing topological models of the accessible receptor surface, for selecting the most relevant mAbs for therapeutic, diagnostic, and research applications and for distinguishing the intellectual property positions of otherwise similar mAbs. While conventional site-directed mutagenesis studies can identify individual amino acid residues that are critical to mAb binding, defining comprehensive epitopes is difficult and time-consuming for these structurally complex proteins. For example, in studies over the past decade, 13 residues (cumulatively) in the GPCR CCR5 have been reported to contribute to the interactions of five well-studied mAbs. 1-5 However, crystallographically defined epitopes contain an average of 20 contact residues each, 6 so these 13 residues likely represent only a portion of all the amino acids that constitute these five epitopes. Because of the importance of CCR5 in HIV infection and inflammation,7 more mAbs have been raised against the native form of this receptor than most other GPCRs, providing a useful set of tools to map its immunodominant structural regions. Here, we have used a high-throughput structure-function analysis strategy, which we refer to as "shotgun mutagenesis", to comprehensively map the critical residues, and in some cases the critical atoms, for these five epitopes of CCR5.To map mAb epitopes, we used an arrayed library of mutations covering nearly all the amino acids in the protein to identify amino acid changes that resulted in loss of mAb reactivity. This approach enabled each epitope to be rapidly mapped within a period of weeks. To create the mutant library, a parental CCR5 plasmid was first created, containing the full length (1059 bp) cDNA for wild type CCR5, flanked by a N-terminal HA epitope tag and a C-terminal V5 epitope tag. Cellular expression of the wild type tagged construct was confirmed by Western blot, immunofluorescence, and flow cytometry. Random mutations were next introduced into the parental CCR5 cDNA using a PCR-based method (Diversify PCR Random Mutagenesis kit, Clontech). Sequenced clones, most exhibiting one to two substitutions, were then selected from these random mutants to create a library with substitutions spanning the entire protein.The final library comprised 734 mutant CCR5 plasmids with substitutions in 346 of the 352 residues of CCR5 (>98% coverage). The average mutation rate per clone was 1.86 amino acids, and each amino acid position was substituted multiple times (an average of 3.95) across the entire library.We used this selective library of CCR5 mutants to map the epitopes of the anti-CCR5 mAbs CTC8, 45523, 45529, 45533 (R&D Systems), and 2D7 (Becton Dickinson). All five mAbs were originally derived, in three independent immunizations, by injecting mice with cells transiently overexpressing human CCR5. 4, 8 These mAbs are therefore representative of the murine immu...

Quantum dots (QDots) are fluorescent semiconductor nanocrystals with a narrow emission spectrum, high quantum yield, and excellent photostability. These unique properties of QDots have been utilized to develop a fluorescent binding assay using biotinylated human T cell leukemia virus type 1 (biot-HTLV-1) conjugated with streptavidin-coated Qdots that enabled both qualitative and quantitative analyses of viral binding. The specificity and linearity of the assay was demonstrated utilizing T cells, the primary HTLV-1-susceptible cell population. Furthermore, differential binding of HTLV-1 was analyzed in additional cell types of clinical relevance including primary CD4 + and CD8 + T cells, dendritic cells (DCs), monocytes, bone marrow progenitor cells, and epithelial cells. DCs exhibited maximum binding affinity when compared to other examined cell types except the Jurkat and SUP-T1 T cell lines. Finally, blocking antibodies directed against a putative HTLV-1 receptor on DCs; DC-SIGN (dendritic cell-specific ICAM-3-grabbing non-integrin), were utilized to study the inhibition of HTLV-1 binding to target cells. Overall, these results demonstrated that this novel high throughput assay can be utilized to study the binding of a biotinylated virus and has implications for screening of viral binding inhibitors as well as host membrane proteins that may serve as receptors for viral entry. KeywordsHTLV-1; quantum dot; viral binding assay 1.IntroductionViral binding and attachment to a host cell membrane, while seemingly simplistic, is a complex area of research for a wide range of viruses. It is often viewed as the first step in infection, whereby a virion is able to attach to a target cell, fuse to the cell membrane, and deliver the contents of the capsid to the cytoplasm of the newly infected cell. The exact mechanism of binding to a host cell varies between viruses and is usually determined by the composition of attachment proteins located within the viral and cellular membranes. The population of cells † A part of this work was presented during 7th International Symposium on NeuroVirology (ISNV), Philadelphia, PA, USA, May 30 - Street, Philadelphia, PA 19102, USA, Tel.: 215-762-8586; Fax: 215-762-1955, E-mail: pjain@drexelmed.edu, Web site: http://www.drexelmed.edu/ Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. (Dhawan et al., 1991;Inghirami et al., 1988). Occasionally, radioactive labels are also utilized for the quantitative estimation of binding (Hubbard, 2003). However, organic fluorophores conventionally used for labeling nucleotides and proteins have poor pho...

Human T-cell leukemia virus type 1 (HTLV-1) is etiologically linked to adult T-cell leukemia and a progressive demyelinating disorder termed HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). One of the most striking features of the immune response in HAM/TSP centers on the expansion of HTLV-1-specific CD8(+) cytotoxic T lymphocyte (CTL) compartment in the peripheral blood and cerebrospinal fluid. More than 90% of the HTLV-1-specific CTLs are directed against the viral Tax (11-19) peptide implying that Tax is available for immune recognition by antigen presenting cells, such as dendritic cells (DCs). DCs obtained from HAM/TSP patients have been shown to be infected with HTLV-1 and exhibit rapid maturation. Therefore, we hypothesized that presentation of Tax peptides by activated DCs to naIve CD8(+) T cells may play an important role in the induction of a Tax-specific CTL response and neurologic dysfunction. In this study, a pathway-specific antigen presenting cell gene array was used to study transcriptional changes induced by exposure of monocyte-derived DCs to extracellular HTLV-1 Tax protein. Approximately 100 genes were differentially expressed including genes encoding toll-like receptors, cell surface receptors, proteins involved in antigen uptake and presentation and adhesion molecules. The differential regulation of chemokines and cytokines characteristic of functional DC activation was also observed by the gene array analyses. Furthermore, the expression pattern of signal transduction genes was also significantly altered. These results have suggested that Tax-mediated DC gene regulation might play a critical role in cellular activation and the mechanisms resulting in HTLV-1-induced disease.