Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

AbstractHybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen

<p>Abstract</p> <p>Background</p> <p>We have been studying the native autoimmune response to cancer through the isolation of human monoclonal antibodies that are cancer specific from cancer patients. To facilitate this work we previously developed a fusion partner cell line for human lymphocytes, MFP-2, that fuses efficiently with both human lymph node lymphocytes and peripheral blood lymphocytes. Using this unique trioma fusion partner cell line we isolated a panel of autologous human monoclonal antibodies, from both peripheral blood and lymph node lymphocytes, which are representative of the native repertoire of anti-cancer specific antibodies from breast cancer patients.</p> <p>Methods</p> <p>The current study employs immunocytochemistry, immunohistochemistry, Western blot analysis as well as Northern blots, Scatchard binding studies and finally SEREX analysis for target antigen identification.</p> <p>Results</p> <p>By application of an expression cloning technique known as SEREX, we determined that the target antigen for two monoclonal antibodies, 27.B1 and 27.F7, derived from lymph node B-cells of a breast cancer patient, is the PDZ domain-containing protein known as GIPC1. This protein is highly expressed not only in cultured human breast cancer cells, but also in primary and metastatic tumor tissues and its overexpression appears to be cancer cell specific. Confocal microscopy revealed cell membrane and cytoplasmic localization of the target protein, which is consistent with previous studies of this protein.</p> <p>Conclusion</p> <p>We have determined that GIPC1 is a novel breast cancer-associated immunogenic antigen that is overexpressed in breast cancer. Its role, however, in the initiation and/or progression of breast cancer remains unclear and needs further clarification.</p

Insights into the intracellular localization, protein associations and artemisinin resistance properties of Plasmodium falciparum K13

The emergence of artemisinin (ART) resistance in Plasmodium falciparum intra-erythrocytic parasites has led to increasing treatment failure rates with first-line ART-based combination therapies in Southeast Asia. Decreased parasite susceptibility is caused by K13 mutations, which are associated clinically with delayed parasite clearance in patients and in vitro with an enhanced ability of ring-stage parasites to survive brief exposure to the active ART metabolite dihydroartemisinin. Herein, we describe a panel of K13-specific monoclonal antibodies and gene-edited parasite lines co-expressing epitope-tagged versions of K13 in trans. By applying an analytical quantitative imaging pipeline, we localize K13 to the parasite endoplasmic reticulum, Rab-positive vesicles, and sites adjacent to cytostomes. These latter structures form at the parasite plasma membrane and traffic hemoglobin to the digestive vacuole wherein artemisinin-activating heme moieties are released. We also provide evidence of K13 partially localizing near the parasite mitochondria upon treatment with dihydroartemisinin. Immunoprecipitation data generated with K13-specific monoclonal antibodies identify multiple putative K13-associated proteins, including endoplasmic reticulum-resident molecules, mitochondrial proteins, and Rab GTPases, in both K13 mutant and wild-type isogenic lines. We also find that mutant K13-mediated resistance is reversed upon co-expression of wild-type or mutant K13. These data help define the biological properties of K13 and its role in mediating P. falciparum resistance to ART treatment

Native human autoantibodies targeting GIPC1 identify differential expression in malignant tumors of the breast and ovary

<p>Abstract</p> <p>Background</p> <p>We have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue.</p> <p>Methods</p> <p>The current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples.</p> <p>Results</p> <p>By screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line). To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors.</p> <p>Conclusion</p> <p>The present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein plays in malignant diseases. In addition, this study suggests that human monoclonal antibodies 27.F7 and 27.B1 should be further evaluated as potential diagnostic tools.</p

Human monoclonal antibodies to West Nile virus identify epitopes on the prM protein

AbstractHybridoma cell lines (2E8, 8G8 and 5G12) producing fully human monoclonal antibodies (hMAbs) specific for the pre-membrane (prM) protein of West Nile virus (WNV) were prepared using a human fusion partner cell line, MFP-2, and human peripheral blood lymphocytes from a blood donor diagnosed with WNV fever in 2004. Using site-directed mutagenesis of a WNV-like particle (VLP) we identified 4 amino acid residues in the prM protein unique to WNV and important in the binding of these hMAbs to the VLP. Residues V19 and L33 are important epitopes for the binding of all three hMAbs. Mutations at residue, T20 and T24 affected the binding of hMAbs, 8G8 and 5G12 only. These hMAbs did not significantly protect AG129 interferon-deficient mice or Swiss Webster outbred mice from WNV infection

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen-4

S analyzed by confocal microscopy and indicates that the target antigen is present in the membrane and cytoplasm. Staining of human breast cancer tissue was analyzed by standard fluorescent microscopy.<p><b>Copyright information:</b></p><p>Taken from "A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen"</p><p>http://www.biomedcentral.com/1471-2407/8/248</p><p>BMC Cancer 2008;8():248-248.</p><p>Published online 24 Aug 2008</p><p>PMCID:PMC2529336.</p><p></p

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen-2

This suggests that two populations of GIPC1 molecules exist in these cells and may be related to the protein doublet identified by Western blot analysis in Figure 2.<p><b>Copyright information:</b></p><p>Taken from "A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen"</p><p>http://www.biomedcentral.com/1471-2407/8/248</p><p>BMC Cancer 2008;8():248-248.</p><p>Published online 24 Aug 2008</p><p>PMCID:PMC2529336.</p><p></p

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen-3

St epithelium cell line HBL100 and breast cancer cell lines MCF-7, T47D, SK-BR-3, MDA231, MDA157 and MDA453. A probe for the GAPDH gene was used to normalize expression. Panel B: Densitometry analysis of the Northern blot was performed to quantitate the mRNA expression. The data indicates that the GIPC1 gene is upregulated in breast cancer cell lines.<p><b>Copyright information:</b></p><p>Taken from "A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen"</p><p>http://www.biomedcentral.com/1471-2407/8/248</p><p>BMC Cancer 2008;8():248-248.</p><p>Published online 24 Aug 2008</p><p>PMCID:PMC2529336.</p><p></p

A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen-1

displayed. Human Ig H and L chains are present in the tissue and are recognized by the secondary anti-human antiserum. The target antigen is detected as a doublet in both the breast cancer tissue and SK-BR-3 cell line but is not detected in normal breast tissue. Both bands in the doublet are present in all breast cancer cell lines analyzed by Western blot but their intensity is variable. B. Immunoblotting with 27.B1 antibody of total cell lysates prepared from SK-BR-3 cells. 27.B1 antibody was preincubated with recombinant GIPC1 protein prior to blotting (lane 1), and compared to non-preincubated control (lane 2).<p><b>Copyright information:</b></p><p>Taken from "A human monoclonal autoantibody to breast cancer identifies the PDZ domain containing protein GIPC1 as a novel breast cancer-associated antigen"</p><p>http://www.biomedcentral.com/1471-2407/8/248</p><p>BMC Cancer 2008;8():248-248.</p><p>Published online 24 Aug 2008</p><p>PMCID:PMC2529336.</p><p></p

Rapid and Flexible Platform To Assess Anti-SARS-CoV-2 Antibody Neutralization and Spike Protein-Specific Antivirals

International audienceThe COVID-19 pandemic caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is ongoing and has shown the community that flexible methods for rapidly identifying and screening candidate antivirals are needed. Assessing virus-neutralizing activity of human serum to monitor population immunity and response to infection and vaccination is key to pandemic control. We developed a virus neutralization platform strategy that relies only on bioinformatic and genetic information of the virus of interest. The platform uses viral envelope glycoprotein cDNAs to set up an assay that mimics multicycle infection but is safe and, therefore, amenable to biosafety level 2 (BSL2) conditions for viruses that require BSL3 facilities (e.g., SARS-CoV-1 and SARS-CoV-2). As a complement to this platform, we present a new cell-based immunofluorescent (CBI) assay that uses SARS-CoV-2 spike protein (S)-expressing cells to accurately measure the neutralization potential of human sera and is readily adaptable to variants of concern. These methods should be useful additions to the tools for assessing antiviral immunity, whether acquired via natural infection or vaccines