Correction: Development of a Humanized Antibody with High Therapeutic Potential against Dengue Virus Type 2.

[This corrects the article DOI: 10.1371/journal.pntd.0001636.].

Effects of PEGylation on capture of dextran-coated magnetic nanoparticles in microcirculation.

BACKGROUND: Magnetic nanoparticles (MNPs) can be localized against hemodynamic forces in blood vessels with the application of an external magnetic field. In addition, PEGylation of nanoparticles may increase the half-life of nanocomposites in circulation. In this work, we examined the effect of PEGylation on the magnetic capture of MNPs in vivo. METHODS: Laser speckle contrast imaging and capillaroscopy were used to assess the magnetic capture of dextran-coated MNPs and red blood cell (RBC) flow in cremaster microvessels of anesthetized rats. Magnetic capture of MNPs in serum flow was visualized with an in vitro circulating system. The effect of PEGylation on MNP-endothelial cell interaction was studied in cultured cells using an iron assay. RESULTS: In microcirculation through cremaster muscle, magnet-induced retention of 250 nm MNPs was associated with a variable reduction in RBC flow, suggesting a dynamic coupling of hemodynamic and magnetic forces. After magnet removal, faster restoration of flow was observed in PEG(+) than PEG(-) group, which may be attributed to a reduced interaction with vascular endothelium. However, PEGylation appears to be required for magnetic capture of 50 nm MNPs in microvessels, which was associated with increased hydrodynamic diameter to 130±6 nm in serum, but independent of the ς-potential. CONCLUSION: These results suggest that PEGylation may enhance magnetic capture of smaller MNPs and dispersion of larger MNPs after magnet removal, which may potentially affect the targeting, pharmacokinetics and therapeutic efficacy.

Generation and Characterization of Antinonstructural Protein 1 Monoclonal Antibodies and Development of Diagnostics for Dengue Virus Serotype 2.

Dengue virus (DENV) circulates in tropical and subtropical areas around the world, where it causes high morbidity and mortality. There is no effective treatment of infection, with supportive care being the only option. Furthermore, early detection and diagnosis are important to facilitate clinical decisions. In this study, seven monoclonal antibodies (mAbs) recognizing nonstructural protein 1 (NS1) of DENV were generated by hybridoma techniques. These antibodies can be divided into two groups: serotype-specific (DB6-1, DB12-3, and DB38-1) and nonspecific (consisting of antibodies DB16-1, DB20-6, DB29-1, and DB41-2). The B-cell epitopes of DB20-6 and DB29-1 were identified by phage display and site-directed mutagenesis, and its binding motif, WXXWGK, was revealed to correspond to amino acid residues 115-120 of the DENV-2 NS1 protein. A diagnostic platform, consisting of a serotype-specific capture antibody and a complex detection antibody, exhibited a detection limit of about 1 ng/mL, which is sufficient to detect NS1 in clinical serum samples from dengue patients. This diagnostic platform displayed better specificity and sensitivity than two examined commercial NS1 diagnostic platforms. In summary, our results indicate that these newly generated mAbs are suitable for detection of NS1 protein of DENV-2 in clinical samples.

Establishment and Comparison of Two Different Diagnostic Platforms for Detection of DENV1 NS1 Protein.

Dengue virus (DENV) infection is currently at pandemic levels, with populations in tropical and subtropical regions at greatest risk of infection. Early diagnosis and management remain the cornerstone for good clinical outcomes, thus efficient and accurate diagnostic technology in the early stage of the disease is urgently needed. Serotype-specific monoclonal antibodies (mAbs) against the DENV1 nonstructural protein 1 (NS1), DA12-4, DA13-2, and DA15-3, which were recently generated using the hybridoma technique, are suitable for use in diagnostic platforms. Immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA) and Western blot analysis further confirmed the serotype specificity of these three monoclonal antibodies. The ELISA-based diagnostic platform was established using the combination of two highly sensitive mAbs (DA15-3 and DB20-6). The same combination was also used for the flow cytometry-based diagnostic platform. We report here the detection limits of flow cytometry-based and ELISA-based diagnostic platforms using these mAbs to be 0.1 and 1 ng/mL, respectively. The collected clinical patient serum samples were also assayed by these two serotyping diagnostic platforms. The sensitivity and specificity for detecting NS1 protein of DENV1 are 90% and 96%, respectively. The accuracy of our platform for testing clinical samples is more advanced than that of the two commercial NS1 diagnostic platforms. In conclusion, our platforms are suitable for the early detection of NS1 protein in DENV1 infected patients.

Generation of Monoclonal Antibodies against Dengue Virus Type 4 and Identification of Enhancing Epitopes on Envelope Protein.

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.

Targeted drug delivery systems mediated by a novel Peptide in breast cancer therapy and imaging.

Targeted delivery of drugs to tumors represents a significant advance in cancer diagnosis and therapy. Therefore, development of novel tumor-specific ligands or pharmaceutical nanocarriers is highly desirable. In this study, we utilized phage display to identify a new targeting peptide, SP90, which specifically binds to breast cancer cells, and recognizes tumor tissues from breast cancer patients. We used confocal and electron microscopy to reveal that conjugation of SP90 with liposomes enables efficient delivery of drugs into cancer cells through endocytosis. Furthermore, in vivo fluorescent imaging demonstrated that SP90-conjugated quantum dots possess tumor-targeting properties. In tumor xenograft and orthotopic models, SP90-conjugated liposomal doxorubicin was found to improve the therapeutic index of the chemotherapeutic drug by selectively increasing its accumulation in tumors. We conclude that the targeting peptide SP90 has significant potential in improving the clinical benefits of chemotherapy in the treatment and the diagnosis of breast cancer.

Development of a humanized antibody with high therapeutic potential against dengue virus type 2.

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.

Molecular mimicry of human endothelial cell antigen by autoantibodies to nonstructural protein 1 of dengue virus.

The pathogenesis of dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), both serious complications of dengue virus (DV) infection, remains unclear. In this study, we found that anti-DV NS1 (nonstructural protein 1) polyclonal antibodies cross-reacted with human umbilical vein endothelial cells (HUVECs). We further identified a complex-specific mAb, DB16-1, which could recognize DV NS1 and cross-react with HUVECs and human blood vessels. The target protein of DB16-1 was further purified by immunoaffinity chromatography. LC-MS/MS analysis and co-immunoprecipitation revealed that the target protein of DB16-1 was human LYRIC (lysine-rich CEACAM1 co-isolated). Our newly generated anti-LYRIC mAbs bound to HUVECs in a pattern similar to that of DB16-1. The B-cell epitope of DB16-1 displayed a consensus motif, Lys-X-Trp-Gly (KXWG), which corresponded to amino acid residues 116-119 of DV NS1 and mimicked amino acid residues 334-337 in LYRIC. Moreover, the binding activity of DB16-1 in NS1 of DV-2 and in LYRIC disappeared after the KXWG epitope was deleted in each. In conclusion, DB16-1 targeted the same epitope in DV NS1 and LYRIC protein on human endothelial cells, suggesting that it might play a role in the pathogenesis of DHF/DSS. Future studies on the role of the anti-NS1 antibody in causing vascular permeability will undoubtedly be performed on sera collected from individuals before, during, and after the endothelial cell malfunction phase of a dengue illness.

DNA methylation and histone modification regulate silencing of OPG during tumor progression.

The identification of molecules that are down-regulated in malignant phenotype is important for understanding tumor biology and their role in tumor suppression. We compared the expression profile of four normal nasal mucosal (NNM) epithelia and a series of nasopharyngeal cancinoma (NPC) cell lines using cDNA microarray and confirmed the actual expression of the selected genes, and found osteoprotegerin (OPG) to be ubiquitously deficient in NPC cells. We also found OPG to be down-regulated in various cancer cell lines, including oral, cervical, ovarian, lung, breast, pancreas, colon, renal, prostate cancer, and hepatoma. Administration of recombinant OPG (rOPG) brought about a reduction in cancer cell growth through apoptotic mechanism. We generated eleven monoclonal antibodies (MAbs) against OPG to study OPG's expression and biological functions in cancer cells. OPG was detected in the tumor stromal regions, but not in the cancer cell per se in surgical specimens of liver cancer. Quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) revealed that OPG was down-regulated in NPC tissues compared with normal nasal polyp (NNP) tissues. In addition, we showed OPG silencing to be associated with promoter methylation as well as histone modifications. In OPG-silenced cancer cell lines, the OPG gene promoter CpG dinucleotides were highly methylated. Compared to normal cells, silenced OPG gene in cancer cells were found to have reduced histone 3 lysine 4 tri-methylation (H3K4me3) and increased histone 3 lysine 27 tri-methylation (H3K27me3). Taken together, these results suggest that OPG silencing in carcinoma cancer cells occurs through epigenetic repression.

Antiangiogenic targeting liposomes increase therapeutic efficacy for solid tumors.

It is known that solid tumors recruit new blood vessels to support tumor growth, but the molecular diversity of receptors in tumor angiogenic vessels might also be used clinically to develop better targeted therapy. In vivo phage display was used to identify peptides that specifically target tumor blood vessels. Several novel peptides were identified as being able to recognize tumor vasculature but not normal blood vessels in severe combined immunodeficiency (SCID) mice bearing human tumors. These tumor-homing peptides also bound to blood vessels in surgical specimens of various human cancers. The peptide-linked liposomes containing fluorescent substance were capable of translocating across the plasma membrane through endocytosis. With the conjugation of peptides and liposomal doxorubicin, the targeted drug delivery systems enhanced the therapeutic efficacy of the chemotherapeutic agent against human cancer xenografts by decreasing tumor angiogenesis and increasing cancer cell apoptosis. Furthermore, the peptide-mediated targeting liposomes improved the pharmacokinetics and pharmacodynamics of the drug they delivered compared with nontargeting liposomes or free drugs. Our results indicate that the tumor-homing peptides can be used specifically target tumor vasculature and have the potential to improve the systemic treatment of patients with solid tumors.

Disease-specific B Cell epitopes for serum antibodies from patients with severe acute respiratory syndrome (SARS) and serologic detection of SARS antibodies by epitope-based peptide antigens.

Severe acute respiratory syndrome (SARS) has emerged as a highly contagious, sometimes fatal disease. To find disease-specific B cell epitopes, phage-displayed random peptide libraries were panned on serum immunoglobulin (Ig) G antibodies from patients with SARS. Forty-nine immunopositive phage clones that bound specifically to serum from patients with SARS were selected. These phageborne peptides had 4 consensus motifs, of which 2 corresponded to amino acid sequences reported for SARS-associated coronavirus (SARS-CoV). Synthetic peptide binding and competitive-inhibition assays further confirmed that patients with SARS generated antibodies against SARS-CoV. Immunopositive phage clones and epitope-based peptide antigens demonstrated clinical diagnostic potential by reacting with serum from patients with SARS. Antibody-response kinetics were evaluated in 4 patients with SARS, and production of IgM, IgG, and IgA were documented as part of the immune response. In conclusion, B cell epitopes of SARS corresponded to novel coronavirus. Our epitope-based serologic test may be useful in laboratory detection of the virus and in further study of the pathogenesis of SARS.

Identification of a dengue virus type 2 (DEN-2) serotype-specific B-cell epitope and detection of DEN-2-immunized animal serum samples using an epitope-based peptide antigen.

In this study, a serotype-specific monoclonal antibody (mAb), D(2) 16-1 (Ab4), against dengue virus type 2 (DEN-2) was generated. The specificity of Ab4, which recognized DEN-2 non-structural protein 1, was determined by ELISA, immunofluorescence and immunoblotting analyses. The serotype-specific B-cell epitope of Ab4 was identified further from a random phage-displayed peptide library; selected phage clones reacted specifically with Ab4 and did not react with other mAbs. Immunopositive phage clones displayed a consensus motif, His-Arg/Lys-Leu/Ile, and a synthetic peptide corresponding to the phage-displayed peptide bound specifically to Ab4. The His and Arg residues in this epitope were found to be crucial for peptide binding to Ab4 and binding activity decreased dramatically when these residues were changed to Leu. The epitope-based synthetic peptide not only identified serum samples from DEN-2-immunized mice and rabbits by ELISA but also differentiated clearly between serum samples from DEN-2- and Japanese encephalitis virus-immunized mice. This mAb and its epitope-based peptide antigen will be useful for serologic diagnosis of DEN-2 infection. Furthermore, DEN-2 epitope identification makes it feasible to dissect antibody responses to DEN and to address the role of antibodies in the pathogenesis of primary and secondary DEN-2 infections.