Novel anti‐dengue monoclonal antibody recognizing conformational structure of the prM‐E heterodimeric complex of dengue virus

Puttikhunt, Chunya; Keelapang, Poonsook; Khemnu, Nuanpan; Sittisombut, Nopporn; Kasinrerk, Watchara; Malasit, Prida

doi:10.1002/jmv.21047

Cited by 17 publications

(18 citation statements)

References 30 publications

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“…Alkaline phosphatase (ALP)-conjugated anti-human IgG (A9544), biotin-conjugated anti-human IgG (B1140), Biotin-conjugated anti-human IgM (B1265) and ALP-streptavidin (S2890) were from Sigma. Mouse monoclonal anti-DENV E (4G2) and anti-DENV prM (1H10) were gifts from AFRIMS, C. Puttikhunt and W. Kasinrerk 45 . Anti-DENV NS3 (E1D8) was a gift from E. Harris.…”

Section: Methodsmentioning

confidence: 99%

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

et al. 2014

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Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

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Section: Methodsmentioning

confidence: 99%

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

et al. 2014

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“…Cells were washed incubated with blocking solution (1% bovine serum albumin/BSA, 0.1% azide and 5% human inactivated plasma in PBS) for 30 min/4ºC. After washing, cells were labeled with mouse monoclonal antibody anti-Dengue Complex (1:100 dilutions, Millipore cat # MAB8705, clone D3-2H2-9-21) (Puttikhunt et al 2008) or isotype (1:100 dilution, cat# MABC004) and anti-mouse IgG Alexa Fluor® 488 (1:400 dilution, Life Technologies) for 60 min/4ºC. Cells were washed with 0.15% saponin, fixed with 2% paraformaldehyde (Sigma) and 2% FBS for 15 min/RT, washed and re-suspended in PBS.…”

Section: Methodsmentioning

confidence: 99%

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

Mello

Valente

Wolff

et al. 2017

Mem. Inst. Oswaldo Cruz

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BACKGROUND Dengue fever may present hemorrhages and cavitary effusions as result of exacerbated immune responses. We investigated hydro-alcoholic extracts from leaves (UGL) and bark (UGB) of the medicinal species Uncaria guinanensis with respect to antiviral effects in Dengue virus (DENV) infection and in immunological parameters associated with in vivo physiopathological features.METHODS Chemical profiles from UGB or UGL were compared in thin layer chromatography and 1H nuclear magnetic resonance using flavonoid compounds and a pentacyclic oxindole alkaloid-enriched fraction as references. DENV-2-infected hepatocytes (Huh-7) were treated with extracts. Cell viability, DENV antigens and immunological factors were detected by enzyme-linked immunosorbent assay (ELISA) or flow cytometry.FINDINGS The UGL mainly differed from UGB by selectively containing the flavonoid kaempferitrin. UGB and UGL improved hepatocyte viability. Both extracts reduced intracellular viral antigen and inhibited the secretion of viral non-structural protein (NS1), which is indicative of viral replication. Reduction in secretion of macrophage migration inhibitory factor was achieved by UGB, of interleukin-6 by UGL, and of interleukin-8 by both UGB and UGL. MAINCONCLUSIONS The U. guianensis extracts presented, antiviral and immunomodulatory effects for DENV and possibly a hepatocyte-protective activity. Further studies may be performed to consider these products as potential candidates for the development of an herbal product for the future treatment of dengue.

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“…Anti-DV2 C antibody was produced, purified, and conjugated with biotin by Genscript. Mouse monoclonal antibodies 2E11 (anti DV2 prM-E heterodimer [19]) and prM6.1 (anti DV2 prM [10]) were kindly provided by Chunya Puttikhunt, Watchara Kasinrerk, and Prida Malasit. Mouse hybridomas producing the monoclonal antibody 4G2 anti-DV E were purchased from ATCC (HB-112).…”

Section: Methodsmentioning

confidence: 99%

“…It is known that prM/E heterodimerization is important in flavivirus assembly, and mutations that prevent this association have been shown to reduce the assembly of viral particles (3,26). We used the antibody 2E11, which specifically recognizes DV2 prM/E heterodimers, to address this (19). We transfected Huh7 cells with pcDNA3.1-D2.VLP or pcDNA3.1-D2(E-299F).VLP plasmids and monitored E and prM/E heterodimer expression by immunofluorescence at 2 days posttransfection (Fig.…”

Section: Figmentioning

confidence: 99%

“…(B and C) To verify that the mutant E-Y299F protein heterodimerizes with prM, Huh7 cells were transfected with D2.VLP and D2(E-Y299F).VLP plasmids (DV2 and Y299F, respectively) and then analyzed on day 2 posttransfection for the presence of the prM-E heterodimer. (B) The coverslips were fixed and analyzed by immunofluorescence using antibodies specific for E and for the prM-E heterodimer (19). A representative experiment out of 2 repeats is shown.…”

Section: Mutagenesis Of the Dv2 E Di/diii Linker Impairs Assembly Of mentioning

confidence: 99%

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Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly

Wispelaere

Yang

2012

J Virol

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The dengue virus (DV) envelope (E) protein is important in mediating viral entry and assembly of progeny virus during cellular infection. Domains I and III (DI and DIII, respectively) of the DV E protein are connected by a highly conserved but poorly ordered region, the DI/DIII linker. Although the flexibility of the DI/DIII linker is thought to be important for accommodating the structural rearrangements undergone by the E protein during viral entry, the function of the linker in the DV infectious cycle is not well understood. In this study, we performed site-directed mutagenesis on conserved residues in the DI/DIII linker of the DV2 E protein and showed that the resulting mutations had little or no effect on the entry process but greatly affected virus assembly. Biochemical fractionation and immunofluorescence microscopy experiments performed on infectious virus as well as in a virus-like particle (VLP) system indicate that the DI/DIII linker mutants express the DV structural proteins at the sites of particle assembly near the ER but fail to form infectious particles. This defect is not due to disruption of E's interaction with prM and pr in immature and mature virions, respectively. Serial passaging of the DV2 mutant E-Y299F led to the identification of a mutation in the membrane-proximal stem region of E that fully compensates for the assembly defect of this DI/DIII linker mutant. Together, our results suggest a critical and previously unidentified role for the E protein DI/DIII linker region during the DV2 assembly process. Dengue virus (DV) is a significant human pathogen and the cause of dengue fever and dengue hemorrhagic fever. The four dengue virus serotypes (DV1, DV2, DV3, and DV4) are members of the Flaviviridae family and have a positive-sense RNA genome encoding a single polyprotein. This polyprotein is processed by host-and DV-encoded proteases into 10 proteins: three structural proteins-core (C), premembrane (prM), and envelope (E)-and seven nonstructural proteins-NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. Following RNA replication and translation, the viral RNA is encapsidated by C to form the nucleocapsid that buds at the endoplasmic reticulum (ER) membrane to associate with the prM and E proteins and form an immature DV virion (25). This immature virion then transits through the secretory pathway, where the virion is matured through cleavage of prM into the membrane (M) protein by furin in the trans-Golgi (21, 22). The mature virion then exits the cell and enters new cells via endocytosis. The acidic environment in the host endosome serves as the physiological trigger for a major conformational change in the E protein that leads to the insertion of its fusion loops in the host endosomal membrane (2, 16). This results in fusion of the viral membrane with the host membrane and delivery of the viral genome to the cytoplasm.The DV E protein belongs to the class II fusion proteins, which share a tertiary structure (11). The DV E protein is anchored in the membrane by two transmembrane domains (28)....

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Novel anti‐dengue monoclonal antibody recognizing conformational structure of the prM‐E heterodimeric complex of dengue virus

Cited by 17 publications

References 30 publications

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus

Decrease in Dengue virus-2 infection and reduction of cytokine/chemokine production by Uncaria guianensis in human hepatocyte cell line Huh-7

Mutagenesis of the DI/DIII Linker in Dengue Virus Envelope Protein Impairs Viral Particle Assembly

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