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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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Effect of anti-E mAb 4G2 on the infectious properties of immature WNV particles in vitro and in vivo.(A) P388D1 cells were infected with immature WNV opsonized with increasing concentrations of 4G2 at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. Data are expressed as means of at least two independent experiments performed in duplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01. (B) Immature WNV was incubated with different concentrations of anti-E 4G2 for 1 hr at 37°C, and injected in mice. A total of 3.4×107 GCPs were given per mouse. Five mice were used for each experimental condition.
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pone-0029957-g005: Effect of anti-E mAb 4G2 on the infectious properties of immature WNV particles in vitro and in vivo.(A) P388D1 cells were infected with immature WNV opsonized with increasing concentrations of 4G2 at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. Data are expressed as means of at least two independent experiments performed in duplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01. (B) Immature WNV was incubated with different concentrations of anti-E 4G2 for 1 hr at 37°C, and injected in mice. A total of 3.4×107 GCPs were given per mouse. Five mice were used for each experimental condition.

Mentions: Because wild type mice do not support DENV replication, and thus are not a suitable animal model for pathogenesis, we instead tested the significance of our findings using the well-established C57BL/6 mouse model of WNV infection [43]. Immature WNV was produced on LoVo cells, as described before [21], [44]. Consistent with our published results, the specific infectivity of the immature WNV preparation was reduced ∼30,000-fold compared to that of st virus prepared in BHK21 cells. Next, we evaluated the infectious properties of immature WNV opsonized with the flavivirus cross-reactive anti-E mAb 4G2 in P388D1 cells at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. In agreement with our data obtained for DENV (Fig. 1C), 4G2 promoted viral infectivity of immature WNV (Fig. 5A). Subsequently, mice were inoculated by intraperitoneal (IP) injection with immature WNV particles with and without prior opsonization with 4G2. Five mice were used for each experimental condition, and 3.4×107 GCPs were inoculated per mouse (based on 104 infectious units for st virus preparations). Mice injected with immature WNV in the absence of Abs showed no clinical signs of infection or lethality, confirming that immature WNV by itself is not infectious (Fig. 5B). In comparison, immature WNV opsonized with anti-E mAb 4G2 became infectious in mice. At a mAb concentration of 4 ng/ml 3 out of 5 mice died, at 40 and 400 ng/ml all mice died and at a 4G2 concentration of 4000 ng/ml 4 out of 5 mice died (Fig. 5B). These results demonstrate that an anti-E mAb directed against DI/II can render immature WNV infectious in vivo in a dose-dependent manner.


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)

Effect of anti-E mAb 4G2 on the infectious properties of immature WNV particles in vitro and in vivo.(A) P388D1 cells were infected with immature WNV opsonized with increasing concentrations of 4G2 at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. Data are expressed as means of at least two independent experiments performed in duplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01. (B) Immature WNV was incubated with different concentrations of anti-E 4G2 for 1 hr at 37°C, and injected in mice. A total of 3.4×107 GCPs were given per mouse. Five mice were used for each experimental condition.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3303773&req=5

pone-0029957-g005: Effect of anti-E mAb 4G2 on the infectious properties of immature WNV particles in vitro and in vivo.(A) P388D1 cells were infected with immature WNV opsonized with increasing concentrations of 4G2 at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. Data are expressed as means of at least two independent experiments performed in duplicate. The error bars represent standard deviations (SD); (n.d.) denotes “not detectable”. Student's t-tests were used to determine significance; *, P<0.01. (B) Immature WNV was incubated with different concentrations of anti-E 4G2 for 1 hr at 37°C, and injected in mice. A total of 3.4×107 GCPs were given per mouse. Five mice were used for each experimental condition.
Mentions: Because wild type mice do not support DENV replication, and thus are not a suitable animal model for pathogenesis, we instead tested the significance of our findings using the well-established C57BL/6 mouse model of WNV infection [43]. Immature WNV was produced on LoVo cells, as described before [21], [44]. Consistent with our published results, the specific infectivity of the immature WNV preparation was reduced ∼30,000-fold compared to that of st virus prepared in BHK21 cells. Next, we evaluated the infectious properties of immature WNV opsonized with the flavivirus cross-reactive anti-E mAb 4G2 in P388D1 cells at MOG 10. At 26 hpi, the supernatant was harvested and virus production was analyzed by plaque assay on BHK21-15 cells. In agreement with our data obtained for DENV (Fig. 1C), 4G2 promoted viral infectivity of immature WNV (Fig. 5A). Subsequently, mice were inoculated by intraperitoneal (IP) injection with immature WNV particles with and without prior opsonization with 4G2. Five mice were used for each experimental condition, and 3.4×107 GCPs were inoculated per mouse (based on 104 infectious units for st virus preparations). Mice injected with immature WNV in the absence of Abs showed no clinical signs of infection or lethality, confirming that immature WNV by itself is not infectious (Fig. 5B). In comparison, immature WNV opsonized with anti-E mAb 4G2 became infectious in mice. At a mAb concentration of 4 ng/ml 3 out of 5 mice died, at 40 and 400 ng/ml all mice died and at a 4G2 concentration of 4000 ng/ml 4 out of 5 mice died (Fig. 5B). These results demonstrate that an anti-E mAb directed against DI/II can render immature WNV infectious in vivo in a dose-dependent manner.

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