Limits...
Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model.

Callaway JB, Smith SA, Widman DG, McKinnon KP, Scholle F, Sempowski GD, Dittmer DP, Crowe JE, de Silva AM, Ting JP - PLoS ONE (2015)

Bottom Line: In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction.Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion.Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America; The Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.

ABSTRACT
Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.

No MeSH data available.


Related in: MedlinePlus

DIV crude supernatant induces rapid IL-1β secretion independent of viral replication.(A-D) Time course of mobilized monocytes inoculated with mock supernatant or DIV crude supernatant with or without mAb 5G22. Cells were washed at 1 hpi and resuspended in fresh medium. Samples were collected at 2, 8, 16, and 24 hpi. (A) Secreted IL-1β. (B) Intracellular DENV E-protein expression. (C) Infectious virus present in the supernatant, as measured by immunoassay on Vero cells. (D) Relative expression of DENV genome copies in mobilized monocytes, measured by real-time PCR. Values are normalized to 2 hpi samples in the absence of mAb 5G22. For A-D, data are pooled from two independent experiments. (E) Secreted IL-1β by mobilized monocytes at 4 hpi. (F) Secreted IL-1β by fresh, non-mobilized monocytes at 4 hpi. (G) Intracellular DENV E-protein expression at 24 hpi with live DIV crude supernatant or DIV crude supernatant inactivated with formalin or UV exposure, all in the presence of mAb 5G22. (H) Secreted IL-1β at 24 hpi using supernatants from 3G. Tests used: Two-Way ANOVA with Dunnett’s post-test (A–D), One-Way ANOVA with Dunnett’s post-test (G, H), Two-Way ANOVA with Tukey’s post-test (E, F). For A and B, gray (A only) and blue asterisks compare DENV and DENV + 5G22, respectively, to mock within each time point. For C and D, blue asterisks compare DENV + 5G22 to the 2 hpi time point.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4547738&req=5

pone.0136708.g003: DIV crude supernatant induces rapid IL-1β secretion independent of viral replication.(A-D) Time course of mobilized monocytes inoculated with mock supernatant or DIV crude supernatant with or without mAb 5G22. Cells were washed at 1 hpi and resuspended in fresh medium. Samples were collected at 2, 8, 16, and 24 hpi. (A) Secreted IL-1β. (B) Intracellular DENV E-protein expression. (C) Infectious virus present in the supernatant, as measured by immunoassay on Vero cells. (D) Relative expression of DENV genome copies in mobilized monocytes, measured by real-time PCR. Values are normalized to 2 hpi samples in the absence of mAb 5G22. For A-D, data are pooled from two independent experiments. (E) Secreted IL-1β by mobilized monocytes at 4 hpi. (F) Secreted IL-1β by fresh, non-mobilized monocytes at 4 hpi. (G) Intracellular DENV E-protein expression at 24 hpi with live DIV crude supernatant or DIV crude supernatant inactivated with formalin or UV exposure, all in the presence of mAb 5G22. (H) Secreted IL-1β at 24 hpi using supernatants from 3G. Tests used: Two-Way ANOVA with Dunnett’s post-test (A–D), One-Way ANOVA with Dunnett’s post-test (G, H), Two-Way ANOVA with Tukey’s post-test (E, F). For A and B, gray (A only) and blue asterisks compare DENV and DENV + 5G22, respectively, to mock within each time point. For C and D, blue asterisks compare DENV + 5G22 to the 2 hpi time point.

Mentions: As enhancing viral replication had no effect on IL-1β secretion, we determined if the kinetics of IL-1β release differ from the kinetics of viral replication. All previous experiments had assessed IL-1β production at 24 hpi, so we initiated a time course with sample collections at 2, 8, 16, and 24 hpi. Mobilized monocytes inoculated with DIV crude supernatant, with or without mAb 5G22, secreted significantly more IL-1β as early as 8 hpi compared to mock conditions (Fig 3A). However, viral replication only began to elevate significantly at 16 hpi (Fig 3B–3D). Intracellular DENV E-protein expression could not be detected until 16 hpi with DIV crude supernatant in the presence of mAb 5G22 (Fig 3B). To further assess replication of the virus, the amount of infectious virus present in the supernatant (Fig 3C) and intracellular presence of DENV genome copies (Fig 3D) were not significantly increased until 24 hpi with DIV crude supernatant and mAb 5G22. Importantly, none of these measures of DENV replication significantly increased when monocytes were inoculated with DIV crude supernatant in the absence of mAb 5G22. These data confirm that IL-1β secretion induced by DIV crude supernatant precedes replication and is independent of ADE.


Source and Purity of Dengue-Viral Preparations Impact Requirement for Enhancing Antibody to Induce Elevated IL-1β Secretion: A Primary Human Monocyte Model.

Callaway JB, Smith SA, Widman DG, McKinnon KP, Scholle F, Sempowski GD, Dittmer DP, Crowe JE, de Silva AM, Ting JP - PLoS ONE (2015)

DIV crude supernatant induces rapid IL-1β secretion independent of viral replication.(A-D) Time course of mobilized monocytes inoculated with mock supernatant or DIV crude supernatant with or without mAb 5G22. Cells were washed at 1 hpi and resuspended in fresh medium. Samples were collected at 2, 8, 16, and 24 hpi. (A) Secreted IL-1β. (B) Intracellular DENV E-protein expression. (C) Infectious virus present in the supernatant, as measured by immunoassay on Vero cells. (D) Relative expression of DENV genome copies in mobilized monocytes, measured by real-time PCR. Values are normalized to 2 hpi samples in the absence of mAb 5G22. For A-D, data are pooled from two independent experiments. (E) Secreted IL-1β by mobilized monocytes at 4 hpi. (F) Secreted IL-1β by fresh, non-mobilized monocytes at 4 hpi. (G) Intracellular DENV E-protein expression at 24 hpi with live DIV crude supernatant or DIV crude supernatant inactivated with formalin or UV exposure, all in the presence of mAb 5G22. (H) Secreted IL-1β at 24 hpi using supernatants from 3G. Tests used: Two-Way ANOVA with Dunnett’s post-test (A–D), One-Way ANOVA with Dunnett’s post-test (G, H), Two-Way ANOVA with Tukey’s post-test (E, F). For A and B, gray (A only) and blue asterisks compare DENV and DENV + 5G22, respectively, to mock within each time point. For C and D, blue asterisks compare DENV + 5G22 to the 2 hpi time point.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4547738&req=5

pone.0136708.g003: DIV crude supernatant induces rapid IL-1β secretion independent of viral replication.(A-D) Time course of mobilized monocytes inoculated with mock supernatant or DIV crude supernatant with or without mAb 5G22. Cells were washed at 1 hpi and resuspended in fresh medium. Samples were collected at 2, 8, 16, and 24 hpi. (A) Secreted IL-1β. (B) Intracellular DENV E-protein expression. (C) Infectious virus present in the supernatant, as measured by immunoassay on Vero cells. (D) Relative expression of DENV genome copies in mobilized monocytes, measured by real-time PCR. Values are normalized to 2 hpi samples in the absence of mAb 5G22. For A-D, data are pooled from two independent experiments. (E) Secreted IL-1β by mobilized monocytes at 4 hpi. (F) Secreted IL-1β by fresh, non-mobilized monocytes at 4 hpi. (G) Intracellular DENV E-protein expression at 24 hpi with live DIV crude supernatant or DIV crude supernatant inactivated with formalin or UV exposure, all in the presence of mAb 5G22. (H) Secreted IL-1β at 24 hpi using supernatants from 3G. Tests used: Two-Way ANOVA with Dunnett’s post-test (A–D), One-Way ANOVA with Dunnett’s post-test (G, H), Two-Way ANOVA with Tukey’s post-test (E, F). For A and B, gray (A only) and blue asterisks compare DENV and DENV + 5G22, respectively, to mock within each time point. For C and D, blue asterisks compare DENV + 5G22 to the 2 hpi time point.
Mentions: As enhancing viral replication had no effect on IL-1β secretion, we determined if the kinetics of IL-1β release differ from the kinetics of viral replication. All previous experiments had assessed IL-1β production at 24 hpi, so we initiated a time course with sample collections at 2, 8, 16, and 24 hpi. Mobilized monocytes inoculated with DIV crude supernatant, with or without mAb 5G22, secreted significantly more IL-1β as early as 8 hpi compared to mock conditions (Fig 3A). However, viral replication only began to elevate significantly at 16 hpi (Fig 3B–3D). Intracellular DENV E-protein expression could not be detected until 16 hpi with DIV crude supernatant in the presence of mAb 5G22 (Fig 3B). To further assess replication of the virus, the amount of infectious virus present in the supernatant (Fig 3C) and intracellular presence of DENV genome copies (Fig 3D) were not significantly increased until 24 hpi with DIV crude supernatant and mAb 5G22. Importantly, none of these measures of DENV replication significantly increased when monocytes were inoculated with DIV crude supernatant in the absence of mAb 5G22. These data confirm that IL-1β secretion induced by DIV crude supernatant precedes replication and is independent of ADE.

Bottom Line: In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction.Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion.Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America; The Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, NC, United States of America.

ABSTRACT
Dengue virus is a major global health threat and can lead to life-threatening hemorrhagic complications due to immune activation and cytokine production. Cross-reactive antibodies to an earlier dengue virus infection are a recognized risk factor for severe disease. These antibodies bind heterologous dengue serotypes and enhance infection into Fc-receptor-bearing cells, a process known as antibody-dependent enhancement of infection. One crucial cytokine seen elevated in severe dengue patients is IL-1β, a potent inflammatory cytokine matured by the inflammasome. We used a highly-physiologic system by studying antibody-dependent enhancement of IL-1β in primary human monocytes with anti-dengue human monoclonal antibodies isolated from patients. Antibody-enhancement increased viral replication in primary human monocytes inoculated with supernatant harvested from Vero cells infected with dengue virus serotype 2 (DENV-2) 16681. Surprisingly, IL-1β secretion induced by infectious supernatant harvested from two independent Vero cell lines was not enhanced by antibody. Secretion of multiple other inflammatory cytokines was also independent of antibody signaling. However, IL-1β secretion did require NLRP3 and caspase-1 activity. Immunodepletion of dengue virions from the infectious supernatant confirmed that virus was not the main IL-1β-inducing agent, suggesting that a supernatant component(s) not associated with the virion induced IL-1β production. We excluded RNA, DNA, contaminating LPS, viral NS1 protein, complement, and cytokines. In contrast, purified Vero-derived DENV-2 16681 exhibited antibody-enhancement of both infection and IL-1β induction. Furthermore, C6/36 mosquito cells did not produce such an inflammatory component, as crude supernatant harvested from insect cells infected with DENV-2 16681 induced antibody-dependent IL-1β secretion. This study indicates that Vero cells infected with DENV-2 16681 may produce inflammatory components during dengue virus propagation that mask the virus-specific immune response. Thus, the choice of host cell and viral purity should be carefully considered, while insect-derived virus represents a system that elicits antibody-dependent cytokine responses to dengue virus with fewer confounding issues.

No MeSH data available.


Related in: MedlinePlus