ABSTRACT
[This corrects the article DOI: 10.1371/journal.pntd.0001636.].
ABSTRACT
The four serotypes of dengue virus (DENV1-4) pose a serious threat to global health. Cross-reactive and non-neutralizing antibodies enhance viral infection, thereby exacerbating the disease via antibody-dependent enhancement (ADE). Studying the epitopes targeted by these enhancing antibodies would improve the immune responses against DENV infection. In order to investigate the roles of antibodies in the pathogenesis of dengue, we generated a panel of 16 new monoclonal antibodies (mAbs) against DENV4. Using plaque reduction neutralization test (PRNT), we examined the neutralizing activity of these mAbs. Furthermore, we used the in vitro and in vivo ADE assay to evaluate the enhancement of DENV infection by mAbs. The results indicate that the cross-reactive and poorly neutralizing mAbs, DD11-4 and DD18-5, strongly enhance DENV1-4 infection of K562 cells and increase mortality in AG129 mice. The epitope residues of these enhancing mAbs were identified using virus-like particle (VLP) mutants. W212 and E26 are the epitope residues of DD11-4 and DD18-5, respectively. In conclusion, we generated and characterized 16 new mAbs against DENV4. DD11-4 and D18-5 possessed non-neutralizing activities and enhanced viral infection. Moreover, we identified the epitope residues of enhancing mAbs on envelope protein. These results may provide useful information for development of safe dengue vaccine.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Antibody-Dependent Enhancement/immunology , Dengue Virus/immunology , Dengue/immunology , Epitopes/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Blocking/immunology , Antibodies, Viral/immunology , Blotting, Western , Dengue/virology , Epitope Mapping , Female , Fluorescent Antibody Technique , Immunoenzyme Techniques , Mice , Mice, Inbred BALB C , Neutralization TestsABSTRACT
Epithelial cell adhesion molecule (EpCAM) is known to be overexpressed in epithelial cancers associated with enhanced malignant potential, particularly colorectal carcinoma (CRC) and head and neck squamous cell carcinoma (HNSCC). However, it is unknown whether progression of malignance can be directly inhibited by targeting EpCAM. Here, we have generated five novel monoclonal antibodies (mAbs) against EpCAM. One of these anti-EpCAM mAbs, EpAb2-6, was found to induce cancer cell apoptosis in vitro, inhibit tumor growth, and prolong the overall survival of both a pancreatic cancer metastatic mouse model and mice with human colon carcinoma xenografts. EpAb2-6 also increases the therapeutic efficacy of irinotecan, fluorouracil, and leucovorin (IFL) therapy in a colon cancer animal model and gemcitabine therapy in a pancreatic cancer animal model. Furthermore, EpAb2-6, which binds to positions Y95 and D96 of the EGF-II/TY domain of EpCAM, inhibits production of EpICD, thereby decreasing its translocation and subsequent signal activation. Collectively, our results indicate that the novel anti-EpCAM mAb can potentially be used for cancer-targeted therapy.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Combined Chemotherapy Protocols/pharmacology , Cell Adhesion Molecules/antagonists & inhibitors , Colonic Neoplasms/drug therapy , Xenograft Model Antitumor Assays , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , Apoptosis/drug effects , Blotting, Western , Camptothecin/administration & dosage , Camptothecin/analogs & derivatives , Cell Adhesion Molecules/immunology , Cell Adhesion Molecules/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Drug Synergism , Epithelial Cell Adhesion Molecule , Flow Cytometry , Fluorouracil/administration & dosage , HCT116 Cells , Humans , Irinotecan , Leucovorin/administration & dosage , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy/methods , Vitamin B Complex/administration & dosageABSTRACT
BACKGROUND: Dengue virus (DENV) is a significant public health threat in tropical and subtropical regions of the world. A therapeutic antibody against the viral envelope (E) protein represents a promising immunotherapy for disease control. METHODOLOGY/PRINCIPAL FINDINGS: We generated seventeen novel mouse monoclonal antibodies (mAbs) with high reactivity against E protein of dengue virus type 2 (DENV-2). The mAbs were further dissected using recombinant E protein domain I-II (E-DI-II) and III (E-DIII) of DENV-2. Using plaque reduction neutralization test (PRNT) and mouse protection assay with lethal doses of DENV-2, we identified four serotype-specific mAbs that had high neutralizing activity against DENV-2 infection. Of the four, E-DIII targeting mAb DB32-6 was the strongest neutralizing mAb against diverse DENV-2 strains. Using phage display and virus-like particles (VLPs) we found that residue K310 in the E-DIII A-strand was key to mAb DB32-6 binding E-DIII. We successfully converted DB32-6 to a humanized version that retained potency for the neutralization of DENV-2 and did not enhance the viral infection. The DB32-6 showed therapeutic efficacy against mortality induced by different strains of DENV-2 in two mouse models even in post-exposure trials. CONCLUSIONS/SIGNIFICANCE: We used novel epitope mapping strategies, by combining phage display with VLPs, to identify the important A-strand epitopes with strong neutralizing activity. This study introduced potential therapeutic antibodies that might be capable of providing broad protection against diverse DENV-2 infections without enhancing activity in humans.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antiviral Agents/therapeutic use , Dengue Virus/immunology , Dengue/immunology , Dengue/therapy , Immunotherapy/methods , Animals , Antibodies, Monoclonal/therapeutic use , Antibodies, Neutralizing/therapeutic use , Antibodies, Viral/therapeutic use , Dengue/virology , Disease Models, Animal , Epitope Mapping , Female , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Survival Analysis , Viral Plaque AssayABSTRACT
An enantiospecific synthesis of Schinzer's ketone 3 from (R)-(+)-pulegone via alpha-carbonyl radical cyclization was accomplished. This work also constitutes an enantiospecific formal syntheses of (-)-pinguisenol and (-)-alpha-pinguisene. The intermediate ketone 4 would be useful for the synthesis of other pinguisane-type sesquiterpenes.
