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Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2.

da Silva Voorham JM, Rodenhuis-Zybert IA, Ayala Nuñez NV, Colpitts TM, van der Ende-Metselaar H, Fikrig E, Diamond MS, Wilschut J, Smit JM - PLoS ONE (2012)

Bottom Line: Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner.The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model.Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands.

ABSTRACT
Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

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Antibodies facilitate binding and internalization of fully immature DENV particles.Binding of immature and standard virion preparations to P388D1 cells with and without prior virus opsonization with antibodies. Virus-cell binding/internalization was measured after 1 h incubation at 37°C by qPCR analysis. Results are shown at conditions of efficient ADE for mAbs that promote viral infectivity. For mAbs that neutralize viral infectivity, a wide antibody concentration range was tested and the condition at which the highest number of GCPs bound per cell is observed is depicted. Data are expressed as means of two independent experiments performed in triplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01.
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pone-0029957-g002: Antibodies facilitate binding and internalization of fully immature DENV particles.Binding of immature and standard virion preparations to P388D1 cells with and without prior virus opsonization with antibodies. Virus-cell binding/internalization was measured after 1 h incubation at 37°C by qPCR analysis. Results are shown at conditions of efficient ADE for mAbs that promote viral infectivity. For mAbs that neutralize viral infectivity, a wide antibody concentration range was tested and the condition at which the highest number of GCPs bound per cell is observed is depicted. Data are expressed as means of two independent experiments performed in triplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01.

Mentions: To investigate if mAbs directed against E, analogous to that seen with enhancing anti-prM antibodies [26], promote uptake of immature DENV particles in cells, we performed a cell binding and internalization assay using qRT-PCR [38]. For this purpose, we selected a subset of enhancing and neutralizing anti-DENV-2 mAbs directed against distinct domains of the E protein. DENV-immune complexes were added to P388D1 cells at a MOG of 1000 and incubated for 1 hr at 37°C. All tested anti-E antibodies facilitated cell binding and internalization of immature particles to levels (3- to 47-fold, *P<0.01) greater than those observed for immature particles in the absence of antibody (Fig. 2). Antibody-opsonized immature particles appeared to bind to cells as efficiently as or even slightly better (mAbs DV2-60, DV2-48 and DV2-53, P<0.01) than st DENV in the absence of antibody. The neutralizing mAbs DV2-30, DV2-40 and DV2-67 bound to a somewhat lower extent to cells than st DENV particles, albeit to comparable levels as the enhancing anti-E mAbs 4G2, DV2-96 and DV2-104. This suggests that neutralization of infection by these mAbs likely occurs in part, at a post-attachment stage after binding of the virus-immune complexes to cells.


Antibodies against the envelope glycoprotein promote infectivity of immature dengue virus serotype 2.

da Silva Voorham JM, Rodenhuis-Zybert IA, Ayala Nuñez NV, Colpitts TM, van der Ende-Metselaar H, Fikrig E, Diamond MS, Wilschut J, Smit JM - PLoS ONE (2012)

Antibodies facilitate binding and internalization of fully immature DENV particles.Binding of immature and standard virion preparations to P388D1 cells with and without prior virus opsonization with antibodies. Virus-cell binding/internalization was measured after 1 h incubation at 37°C by qPCR analysis. Results are shown at conditions of efficient ADE for mAbs that promote viral infectivity. For mAbs that neutralize viral infectivity, a wide antibody concentration range was tested and the condition at which the highest number of GCPs bound per cell is observed is depicted. Data are expressed as means of two independent experiments performed in triplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303773&req=5

pone-0029957-g002: Antibodies facilitate binding and internalization of fully immature DENV particles.Binding of immature and standard virion preparations to P388D1 cells with and without prior virus opsonization with antibodies. Virus-cell binding/internalization was measured after 1 h incubation at 37°C by qPCR analysis. Results are shown at conditions of efficient ADE for mAbs that promote viral infectivity. For mAbs that neutralize viral infectivity, a wide antibody concentration range was tested and the condition at which the highest number of GCPs bound per cell is observed is depicted. Data are expressed as means of two independent experiments performed in triplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01.
Mentions: To investigate if mAbs directed against E, analogous to that seen with enhancing anti-prM antibodies [26], promote uptake of immature DENV particles in cells, we performed a cell binding and internalization assay using qRT-PCR [38]. For this purpose, we selected a subset of enhancing and neutralizing anti-DENV-2 mAbs directed against distinct domains of the E protein. DENV-immune complexes were added to P388D1 cells at a MOG of 1000 and incubated for 1 hr at 37°C. All tested anti-E antibodies facilitated cell binding and internalization of immature particles to levels (3- to 47-fold, *P<0.01) greater than those observed for immature particles in the absence of antibody (Fig. 2). Antibody-opsonized immature particles appeared to bind to cells as efficiently as or even slightly better (mAbs DV2-60, DV2-48 and DV2-53, P<0.01) than st DENV in the absence of antibody. The neutralizing mAbs DV2-30, DV2-40 and DV2-67 bound to a somewhat lower extent to cells than st DENV particles, albeit to comparable levels as the enhancing anti-E mAbs 4G2, DV2-96 and DV2-104. This suggests that neutralization of infection by these mAbs likely occurs in part, at a post-attachment stage after binding of the virus-immune complexes to cells.

Bottom Line: Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner.The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model.Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology, Molecular Virology Section, University Medical Center Groningen and University of Groningen, Groningen, The Netherlands.

ABSTRACT
Cross-reactive dengue virus (DENV) antibodies directed against the envelope (E) and precursor membrane (prM) proteins are believed to contribute to the development of severe dengue disease by facilitating antibody-dependent enhancement of infection. We and others recently demonstrated that anti-prM antibodies render essentially non-infectious immature DENV infectious in Fcγ-receptor-expressing cells. Immature DENV particles are abundantly present in standard (st) virus preparations due to inefficient processing of prM to M during virus maturation. Structural analysis has revealed that the E protein is exposed in immature particles and this prompted us to investigate whether antibodies to E render immature particles infectious. To this end, we analyzed the enhancing properties of 27 anti-E antibodies directed against distinct structural domains. Of these, 23 bound to immature particles, and 15 enhanced infectivity of immature DENV in a furin-dependent manner. The significance of these findings was subsequently tested in vivo using the well-established West Nile virus (WNV) mouse model. Remarkably, mice injected with immature WNV opsonized with anti-E mAbs or immune serum produced a lethal infection in a dose-dependent manner, whereas in the absence of antibody immature WNV virions caused no morbidity or mortality. Furthermore, enhancement infection studies with standard (st) DENV preparations opsonized with anti-E mAbs in the presence or absence of furin inhibitor revealed that prM-containing particles present within st virus preparations contribute to antibody-dependent enhancement of infection. Taken together, our results support the notion that antibodies against the structural proteins prM and E both can promote pathogenesis by enhancing infectivity of prM-containing immature and partially mature flavivirus particles.

Show MeSH
Related in: MedlinePlus