Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1–4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, μMT[superscript −/−] mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen

A Model of DENV-3 Infection That Recapitulates Severe Disease and Highlights the Importance of IFN-γ in Host Resistance to Infection

There are few animal models of dengue infection, especially in immunocompetent mice. Here, we describe alterations found in adult immunocompetent mice inoculated with an adapted Dengue virus (DENV-3) strain. Infection of mice with the adapted DENV-3 caused inoculum-dependent lethality that was preceded by several hematological and biochemical changes and increased virus dissemination, features consistent with severe disease manifestation in humans. IFN-γ expression increased after DENV-3 infection of WT mice and this was preceded by increase in expression of IL-12 and IL-18. In DENV-3-inoculated IFN-γ−/− mice, there was enhanced lethality, which was preceded by severe disease manifestation and virus replication. Lack of IFN-γ production was associated with diminished NO-synthase 2 (NOS2) expression and higher susceptibility of NOS2−/− mice to DENV-3 infection. Therefore, mechanisms of protection to DENV-3 infection rely on IFN-γ-NOS2-NO-dependent control of viral replication and of disease severity, a pathway showed to be relevant for resistance to DENV infection in other experimental and clinical settings. Thus, the model of DENV-3 infection in immunocompetent mice described here represents a significant advance in animal models of severe dengue disease and may provide an important tool to the elucidation of immunopathogenesis of disease and of protective mechanisms associated with infection

Disease parameters in C57BL/6 mice infected with an adapted strain of DENV-3.

<p>(A) WT mice (<i>n</i> = 6 mice per group) were inoculated with different inoculums of adapted-DENV-3 (i.p) and lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. In Figs (B–L) WT mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection mice were culled and blood and tissues were collected for the following analysis: (B) Change in body weight was expressed as percentage of initial weight loss. (C) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (D), hematocrit was expressed as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (E) Changes in vascular permeability in the liver and lungs are shown as µg Evans blue per 100 mg of tissue (left and right panels, respectively). (F) Shows changes in Systolic blood pressure from baseline until day 7 after infection expressed as Δ of blood pressure in mmHg. (G) AST (left panel) and ALT (right panel) activity determination in plasma of control and DENV-3-infected mice was shown as U/dL of plasma. (H–L) Concentrations of IL-6, TNF-α, IFN-γ IL-12/23p40 and IL-18, quantified by ELISA. Results are shown as pg per mL (serum) or pg per 100 mg (tissue). All results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND – not detectable. NA – not assessed. NI- Not-infected. dpi – days post-infection.</p

IFN-γ controls NOS2-mediated NO production during adapted-DENV-3 infection.

<p>(A–C) WT mice (<i>n</i> = 6 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of adapted-DENV-3 (i.p) and 3, 5 or 7 days after infection, mice were culled and tissue were collected for the following analysis: (A) Determination of NOS2 RNA expression by qPCR in spleen of control and DENV-3 infected mice. Results are shown as fold increase over basal expression in naive mice. (B) Determination of NOS2 staining by immunohistochemistry in liver sections of control and DENV-3 infected mice. Results are expressed as number of positive cells per mm² of liver. (C) Esplenocytes were incubated with DAF-2DA and fluorescence determined. Results are expressed as fold increase in fluorescence over stained cells of naive mice. (D–E), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and tissues were collected for the following analysis: (D) Determination of NOS2 RNA expression by qPCR in spleen of WT and IFN-γ^−/− DENV-3 infected mice. Results are shown as fold increase over basal expression in naive mice. (E) Determination of NOS2 staining by immunohistochemistry in liver sections of WT and IFN-γ^−/− DENV-3 infected mice. Results are expressed as number of positive cells per mm² of liver. Results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control naive mice. # for P<0.05 when compared to WT infected mice. 10LD₅₀ corresponds to 1000 PFU of DENV-3. 1LD₅₀ corresponds to 100 PFU of DENV-3. dpi – days post-infection. NI – Not-infected.</p

NOS2^−/− mice are more susceptible to adapted-DENV-3 infection.

<p>(A) WT and NOS2^−/− mice (n = 8 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and lethality was evaluated every 12 hours during 14 days. Results are expressed as % of survival. In (B–J), WT and NOS2^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the seventy day of infection mice were culled and blood and tissues were collected for the following analysis: (B–D) Viral loads were recovered from the blood (B), spleen (C) and liver (D), respectively. Results are shown as the log of PFU per per mL of blood or g of tissue. (E) Serial sections from liver of WT and NOS2^−/− mice were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a (IgG2a data not shown), and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue.Results are expressed as number of NS3-positive hepatocytes. (F) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (G), hematocrit was shown as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (H) Changes in Systolic blood pressure from baseline until day 5 after infection expressed as Δ of blood pressure in mmHg. In (I), AST activity determination in plasma was shown as U/dL of plasma. (J) shows semi-quantitative analysis of hepatic damage (histopathological analysis performed as modified from Paes et al, 2009) and Hematoxylin & Eosin staining of liver sections of control and WT and NOS2^−/− DENV-3-infected mice, seven days after infection. Scale Bar - 400 µm. The images presented are representative of an animal on the seventh day of infection. All results are expressed as mean ± SEM and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. # for P<0.05 when compared to WT infected mice.1LD₅₀ corresponds to 100 PFU of adapted-DENV-3. NI- Not-infected. dpi – days post-infection. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p

IFN-γ production is required for host resistance to adapted-DENV-3 primary infection.

<p>(A) WT mice (<i>n</i> = 4 mice per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and seven days later, mice were culled, and splenic cells isolated for assaying IFN-γ production by cellular staining with labeled antibodies and FACS analysis. Results are expressed as % of IFN-γ-positive cells in each population. (B) WT and IFN-γ^−/− mice (n = 8 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and lethality was evaluated every 12 hours during 14 days. Results are expressed as % of survival. In (C–J), WT and IFN-γ^−/− mice (n = 6 per group) were inoculated with 1LD₅₀ (100 PFU) of DENV-3 (i.p) and in the fifth day of infection mice were culled and blood and tissues were collected for the following analysis: (C–D) Viral loads were recovered from the blood (C), spleen and liver (D, left and right panels), respectively. Results are shown as the log of PFU per mL of blood or per g of tissue. (E) Serial sections from each liver were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a (IgG2a data not shown), and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. Results are expressed as number of NS3-positive hepatocytes. (F) Mechanical hypernociception was assessed daily. Results are shown as the difference between the force (g) necessary to induce dorsal flexion of tibio-tarsal joint, followed by paw withdraw, before and after DENV-3 inoculation. In (G), hematocrit was shown as % volume occupied by red blood cells (left panel) and the number of platelets was shown as platelets ×10³/µl of blood (right panel). (H) Changes in Systolic blood pressure from baseline until day 5 after infection expressed as Δ of blood pressure in mmHg. In (I), AST activity determination in plasma, shown as U/dL of plasma. (J) shows semi-quantitative analysis of hepatic damage (histopathological analysis performed as modified from Paes et al, 2009) and Hematoxylin & Eosin staining of liver sections of control and WT and IFN-γ^−/− DENV-3-infected mice, five days after infection. Scale bars - 400 µm. The images presented are representative of an animal on the fifth day of infection. All results are expressed as mean ± SEM (except for C–D, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. # fo P,0.05 when compared to WT infected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. 1LD₅₀ corresponds to 100 PFU of adapted-DENV-3. ND – not detectable. NI- Not-infected. dpi – days post-infection. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p

Characterization of virologic and histopathological parameters in C57BL/6j mice upon adapted-DENV-3 infection.

<p>(A–D) C57BL/6j mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the third, fifth or in the seventh day of infection, mice were culled and blood and tissues were collected for the following analysis: (A–C) Viral loads were recovered from the spleen, liver and blood, respectively. Results are shown as the log of PFU per g of tissue or per mL of blood. (D) Shows virus NS1 antigen serum levels by ELISA and expressed as O.D. (E–F) C57BL/6j mice (n = 6 per group) were inoculated with 10LD₅₀ (1000 PFU) of DENV-3 (i.p) and in the seventh day of infection mice were culled and liver collected for the following analyses: (E) Liver was collected, formalin-fixed and processed into paraffin sections. Serial sections from each tissue were stained with anti-DV NS3 antibody E1D8 (NS3) or an isotype control mouse IgG2a, and multiple sections of each tissue type were thoroughly examined for staining. Positive staining for NS3 is brown while hematoxylin counterstain is blue. (F) shows semi-quantitative analysis of hepatic damage and Hematoxylin & Eosin staining of liver sections of control and DENV-3-infected mice, seven days after infection (Scale Bar - 400 µm). The images presented are representative of an animal on the seventh day of infection. In (G) Viral inoculum (10LD₅₀ or 1000 PFU) was heat inactivated (Heat, 56°C, 60 min) or treated with UV light (UV, 15 min) before inoculation in C57BL/6j mice. Lethality was evaluated every 12 hours for 14 days. (H) WT mice (<i>n</i> = 6 mice per group) were pretreated i.p with 100 µL of Anti-DENV-3 antiserum or control serum (pre-immune serum) before inoculation of 10LD₅₀ (1000 PFU) of adapted-DENV-3 (i.p). Lethality was evaluated every 12 hours for 14 days. Results are expressed as % of survival. Results are expressed as mean ± SEM (except for A–C, expressed as median) and are representative of at least two experiments. * for P<0.05 when compared to control uninfected mice. 10 LD₅₀ corresponds to 1000 PFU of adapted-DENV-3. ND- not detected. NI- not-infected. dpi- days post-infection. NC – Negative control. HS – hepatocyte swelling. N – necrosis. D – degeneration. H – hemorrhage. OS – Overall Score.</p