scholarly journals Development of Virus-Like-Particle Vaccine and Reporter Assay for Zika Virus

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Himanshu Garg ◽  
Melina Sedano ◽  
Gabrielle Plata ◽  
Erin B. Punke ◽  
Anjali Joshi
Keyword(s):  
Cell Line ◽  
Zika Virus ◽  
Neutralizing Antibody ◽  
Stable Cell Line ◽  
Reporter Virus ◽  
E Proteins ◽  
Virus Particles ◽  
Microneutralization Assay ◽  
Virus Like Particle ◽  
A Cell

ABSTRACT Recent worldwide outbreaks of Zika virus (ZIKV) infection and the lack of an approved vaccine raise serious concerns regarding preparedness to combat this emerging virus. We used a virus-like particle (VLP)-based approach to develop a vaccine and a microneutralization assay for ZIKV. A synthetic capsid-premembrane-envelope (C-prM-E) gene construct of ZIKV was used to generate reporter virus particles (RVPs) that package a green fluorescent protein (GFP) reporter-expressing West Nile virus (WNV) replicon. The assay was adapted to a 96-well format, similar to the plaque reduction neutralization test (PRNT), and showed high reproducibility with specific detection of ZIKV neutralizing antibodies. Furthermore, C-prM-E and prM-E VLPs were tested as vaccine candidates in mice and compared to DNA vaccination. While the ZIKV prM-E construct alone was sufficient for generating VLPs, efficient VLP production from the C-prM-E construct could be achieved in the presence of the WNV NS2B-3 protease, which cleaves C from prM, allowing virus release. Immunization studies in mice showed that VLPs generated higher neutralizing antibody titers than those with the DNA vaccines, with C-prM-E VLPs giving slightly higher titers than those with prM-E VLPs. The superiority of C-prM-E VLPs suggests that inclusion of capsid may have benefits for ZIKV and other flaviviral VLP vaccines. To facilitate the VLP platform, we generated a stable cell line expressing high levels of ZIKV prM-E proteins that constitutively produce VLPs as well as a cell line expressing ZIKV C-prM-E proteins for RVP production. While several vaccine platforms have been proposed for ZIKV, this study describes a safe, effective, and economical VLP-based vaccine against ZIKV. IMPORTANCE To address the growing Zika virus epidemic, we undertook this study with two objectives: first, to develop a safe, effective, and economical vaccine for ZIKV, and second, to develop a rapid and versatile assay to detect the anti-ZIKV immune response. We generated a cell line stably expressing ZIKV prM-E that produces large amounts of VLPs in the supernatant and a ZIKV C-prM-E cell line that produces reporter virus particles upon transfection with a GFP replicon plasmid. The prM-E VLPs induced a strong neutralizing antibody response in mice that was better when the capsid was included. VLP-based vaccines showed significantly better neutralizing antibody responses than those with their DNA counterparts. The RVP-based microneutralization assay worked similarly to the PRNT assay, with a rapid GFP readout in a 96-well format. Our VLP-based platform provides a source for a ZIKV vaccine and diagnosis that can rapidly be adapted to current outbreaks.

Capsid containing virus like particle vaccine against Zika virus made from a stable cell line

Vaccine ◽  
2019 ◽  
Vol 37 (48) ◽  
pp. 7123-7131 ◽  
Author(s):  
Himanshu Garg ◽  
Tugba Mehmetoglu-Gurbuz ◽  
Gregory M. Ruddy ◽  
Anjali Joshi
Keyword(s):  
Cell Line ◽  
Zika Virus ◽  
Stable Cell Line ◽  
Virus Like Particle

Aberrant Type C Virus Production in a Cell Line Derived from Balb/3T3

1972 ◽  
Vol 30 ◽  
pp. 286-287
Author(s):  
N. Savage ◽  
A. Hackett
Keyword(s):  
Electron Microscopy ◽  
Cell Line ◽  
Leukemia Virus ◽  
Morphological Characteristics ◽  
Virus Production ◽  
Oncogenic Viruses ◽  
Endogenous Virus ◽  
Virus Particles ◽  
Moloney Leukemia Virus ◽  
A Cell

A cell line, UC1-B, which was derived from Balb/3T3 cells, maintains the same morphological characteristics of the non-transformed parental culture, and shows no evidence of spontaneous virus production. Survey by electron microscopy shows that the cell line consists of spindle-shaped cells with no unusual features and no endogenous virus particles.UC1-B cells respond to Moloney leukemia virus (MLV) infection by a change in morphology and growth pattern which is typical of cells transformed by sarcoma virus. Electron microscopy shows that the cells are now variable in shape (rounded, rhomboid, and spindle), and each cell type has some microvilli. Virtually all (90%) of the cells show virus particles developing at the cell surface and within the cytoplasm. Maturing viruses, typical of the oncogenic viruses, are found along with atypical tubular forms in the same cell.

Gastric inhibitory polypeptide release from a tumor-derived cell line

1995 ◽  
Vol 269 (2) ◽  
pp. E316-E322 ◽  
Author(s):  
T. J. Kieffer ◽  
Z. Huang ◽  
C. H. McIntosh ◽  
A. M. Buchan ◽  
J. C. Brown ◽  
...  
Keyword(s):  
Cell Line ◽  
High Performance ◽  
Neutralizing Antibody ◽  
Gastric Inhibitory Polypeptide ◽  
Total Cell ◽  
Controlled Environment ◽  
Intestinal Tumors ◽  
Cell Content ◽  
Basal Release ◽  
A Cell

A cell line derived from intestinal tumors of transgenic mice (STC-1) was subcloned to produce a stable line with approximately 30% immunoreactive gastric inhibitory polypeptide (irGIP)-containing cells (STC 6-14). High-performance liquid chromatography (HPLC) of STC 6-14 extracts indicated that the tumor cell-derived irGIP had the same retention time as synthetic porcine GIP-(1-42) (pGIP). Approximately 30% of the cells also contained immunoreactive somatostatin (irSS), which eluted as a single peak on HPLC, corresponding with SS-(1-14). On average, each well of extracted cells (5.0 x 10(5) cultured 4 days) contained 33.3 +/- 1.4 ng irGIP and 18.4 +/- 1.5 ng irSS. Basal release of irGIP in the presence of 5 mM glucose was 733 +/- 58 pg.ml cells-1.2h-1 (2.20 +/- 0.17% of total cell content; TCC) and doubled at 20 mM glucose (4.20 +/- 0.42% TCC). The response to glucose was augmented by addition of a SS neutralizing antibody (SOMA-10) and suppressed by 10 nM SS. Basal release of irSS in 5 mM glucose was 377 +/- 35 pg.ml cells-1.2h-1 (2.05 +/- 0.19% TCC) and was increased by glucose (> or = 15 mM) and the addition of pGIP (> or = 1 nM). The STC 6-14 cell line represents a model to study the synthesis, storage, and release of GIP and SS in a controlled environment.

scholarly journals Establishment of a Stable Cell Line Coexpressing Dengue Virus-2 and Green Fluorescent Protein for Screening of Antiviral Compounds

2011 ◽  
Vol 17 (3) ◽  
pp. 283-292 ◽  
Author(s):  
V. Leardkamolkarn ◽  
W. Sirigulpanit
Keyword(s):  
Green Fluorescent Protein ◽  
Dengue Virus ◽  
Cell Line ◽  
Fluorescent Protein ◽  
Antiviral Drug ◽  
Stable Cell Line ◽  
Entry Site ◽  
Mouth Disease Virus ◽  
A Cell ◽  
Green Fluorescent

This study aimed to generate a stable cell line harboring subgenomic dengue virus replicon and a green fluorescent gene (DENV/GFP) for a cell-based model to screen anti-DENV compounds. The gene-encoding envelope protein of DENV-2 was deleted and then replaced with fragments of the GFP gene and a foot-and-mouth-disease virus 2A–derived cleavage site. The human cytomegalovirus immediate early and antisense hepatitis delta virus ribozyme sequences were added at the 5′- and 3′-ends. An internal ribosome entry site and neomycin resistance genes were placed upstream and next to the NS1 gene. The recombinant plasmids were propagated in a mammalian cell line. A stable cell line with the brightest green fluorescent protein and the highest viral protein and RNA expression was selected from six clones. The clone was then examined for effectiveness in an antiviral drug screening assay with compounds isolated from the local plants using two known antiviral agents as controls. Two novel flavones, PMF and TMF, were discovered having DENV-inhibitory properties. The data were validated by a conventional plaque titration assay. The results indicate that this newly developed cell line is efficient for use as a cell-based model for primary screening of anti-DENV compounds.

scholarly journals An epitope-resurfaced virus-like particle can induce broad neutralizing antibody against four serotypes of dengue virus

2018 ◽  
Author(s):  
Wen-Fan Shen ◽  
Jedhan Ucat Galula ◽  
Jyung-Hurng Liu ◽  
Mei-Ying Liao ◽  
Chang-Hao Huang ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Quaternary Structure ◽  
Neutralizing Antibody ◽  
Icosahedral Symmetry ◽  
E Proteins ◽  
Cell Repertoire ◽  
Virus Like Particles ◽  
B Cell Repertoire ◽  
Protein Dimers ◽  
Virus Like Particle

AbstractDengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein we show for the first time that mD2VLP particles possess a T=1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes through cryo-electron microscopy reconstruction. Mice vaccinated with highly matured virus-like particles derived from DENV serotype 2 (mD2VLP) can generate higher cross reactive (CR) neutralization antibodies (NtAbs) and were protected against all 4 serotypes of DENV through clonal expansion supported by hybridoma and B-cell repertoire analysis. Our results revealed that a “epitope-resurfaced” mature-form dengue VLP has the potential to induce quaternary structure-recognizing broad CR NtAbs.

scholarly journals Establishment of a Cell Model Based on FKBP12 Dimerization for Screening of FK506-like Neurotrophic Small Molecular Compounds

2006 ◽  
Vol 11 (3) ◽  
pp. 225-235
Author(s):  
He Xiao ◽  
Li-Li Wang ◽  
Cui-Ling Shu ◽  
Ming Yu ◽  
Song Li ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Model ◽  
Immunosuppressive Agent ◽  
Stable Cell Line ◽  
Fk506 Binding Protein ◽  
Neurotrophic Activity ◽  
Transplant Patients ◽  
Promote Neurite Outgrowth ◽  
Immunosuppressive Action ◽  
A Cell

FK506 is an efficient immunosuppressive agent with an increasing number of clinical applications. It has been approved to prevent rejection in transplant patients and be efficacious in several autoimmune diseases. Its immunosuppressive activity results from binding to receptor proteins designated as immunophilins (i.e., FKBP12, FK506 binding protein). Recent studies have suggested that FK506 can promote neurite outgrowth as a 2nd activity. Furthermore, it has been shown that the neurotrophic property of FK506 is independent of its immunosuppressive action. Although the mechanism of its neurotrophic activity has not yet been well elucidated, FKBP12 is identified as a drug target, and much effort has been directed toward the design of FKBP12-binding molecules, which are neurotrophic but nonimmunosuppressive, for clinical use. In this present study, the authors constructed a stable cell line, which underwent apoptosis upon treatment by AP20187, a wholly synthesized, cell-permeable dimeric FK506 derivative, based on FKBP12-mBax dimerization. This AP20187-mediated apoptosiswas rapidly reversed by the addition of an FKBP12-binding competitormolecule (FK506 or rapamycin), indicating that this cell line might be used to screen FK506 derivatives. Using the screening model, hundreds of synthetic FK506 analogs were analyzed. A promising compound, named N308, was obtained. The results showed that N308 could inhibit AP20187-induced gene-modified target cell apoptosis and elicit augmentation of neurite extension from both cultured PC-12 cells and chicken dorsal root ganglia cultures.

Immunoelectron Microscopy of Type C Virus Particles Produced in a Cell Line Derived from Pleural Effusion of a Lymphoma Patient

1971 ◽  
Vol 29 ◽  
pp. 374-375
Author(s):  
T. Shigematsu ◽  
E. S. Priori ◽  
L. Dmochowski ◽  
J. R. Wilbur
Keyword(s):  
Pleural Effusion ◽  
Cell Line ◽  
Monolayer Culture ◽  
Human Tumor ◽  
Tumor Cell Line ◽  
Lymphoma Patient ◽  
Human Tumor Cell ◽  
Virus Particles ◽  
A Cell ◽  
Type C

A monolayer culture (ESP-1) was established with cells obtained from pleural effusion of a patient with Burkitt lymphoma (American type). Virus particles, similar to murine type C virus particles, were first observed in cells of the 10th passage of the culture. Mature and immature, including budding, virus particles were frequently found in all subsequent passages of this continuous culture (including 28th).A human tumor cell line continuously producing type C virus particles provides a valuable new system for immunological studies of such virus particles.

scholarly journals Epitope resurfacing on dengue virus-like particle vaccine preparation to induce broad neutralizing antibody

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Wen-Fan Shen ◽  
Jedhan Ucat Galula ◽  
Jyung-Hurng Liu ◽  
Mei-Ying Liao ◽  
Cheng-Hao Huang ◽  
...  
Keyword(s):  
Dengue Virus ◽  
Quaternary Structure ◽  
Neutralizing Antibody ◽  
Icosahedral Symmetry ◽  
E Proteins ◽  
Protein Dimers ◽  
Virus Like Particle ◽  
Arboviral Diseases ◽  
Potential Vaccine ◽  
Microscopy Examination

Dengue fever is caused by four different serotypes of dengue virus (DENV) which is the leading cause of worldwide arboviral diseases in humans. Virus-like particles (VLPs) containing flavivirus prM/E proteins have been demonstrated to be a potential vaccine candidate; however, the structure of dengue VLP is poorly understood. Herein VLP derived from DENV serotype-2 were engineered becoming highly matured (mD2VLP) and showed variable size distribution with diameter of ~31 nm forming the major population under cryo-electron microscopy examination. Furthermore, mD2VLP particles of 31 nm diameter possess a T = 1 icosahedral symmetry with a groove located within the E-protein dimers near the 2-fold vertices that exposed highly overlapping, cryptic neutralizing epitopes. Mice vaccinated with mD2VLP generated higher cross-reactive (CR) neutralization antibodies (NtAbs) and were fully protected against all 4 serotypes of DENV. Our results highlight the potential of ‘epitope-resurfaced’ mature-form D2VLPs in inducing quaternary structure-recognizing broad CR NtAbs to guide future dengue vaccine design.

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