Maternal Antibody-Mediated Disease Enhancement in Type I Interferon-Deficient Mice Leads to Lethal Disease Associated with Liver Damage.

Epidemiological studies have reported that most of the severe dengue cases occur upon a secondary heterologous infection. Furthermore, babies born to dengue immune mothers are at greater risk of developing severe disease upon primary infection with a heterologous or homologous dengue virus (DENV) serotype when maternal antibodies reach sub-neutralizing concentrations. These observations have been explained by the antibody mediated disease enhancement (ADE) phenomenon whereby heterologous antibodies or sub-neutralizing homologous antibodies bind to but fail to neutralize DENV particles, allowing Fc-receptor mediated entry of the virus-antibody complexes into host cells. This eventually results in enhanced viral replication and heightened inflammatory responses. In an attempt to replicate this ADE phenomenon in a mouse model, we previously reported that upon DENV2 infection 5-week old type I and II interferon (IFN) receptors-deficient mice (AG129) born to DENV1-immune mothers displayed enhancement of disease severity characterized by increased virus titers and extensive vascular leakage which eventually led to the animals' death. However, as dengue occurs in immune competent individuals, we sought to reproduce this mouse model in a less immunocompromised background. Here, we report an ADE model that is mediated by maternal antibodies in type I IFN receptor-deficient A129 mice. We show that 5-week old A129 mice born to DENV1-immune mothers succumbed to a DENV2 infection within 4 days that was sub-lethal in mice born to naïve mothers. Clinical manifestations included extensive hepatocyte vacuolation, moderate vascular leakage, lymphopenia, and thrombocytopenia. Anti-TNFα therapy totally protected the mice and correlated with healthy hepatocytes. In contrast, blocking IL-6 did not impact the virus titers or disease outcome. This A129 mouse model of ADE may help dissecting the mechanisms involved in dengue pathogenesis and evaluate the efficacy of vaccine and therapeutic candidates

Anti-TNFα treatment of DENV2-infected mice born to DENV1-immune mothers.

<p>Five to six-weeks old A129 mice born to DENV1-immune mothers were infected iv with 10⁶ PFU of D2Y98P-PP1. At day 2 p.i., the mice were injected iv with 100 μg of anti-TNFα neutralizing antibody (open square) or 100μg of isotype control antibody (black square). (A) Survival rate and (B) mean clinical score of anti-TNFα-treated and isotype control antibody-treated DENV2-infected mice born to DENV1-immune mothers (n = 7–8). (C) Viremia and (D) serum AST levels of anti-TNFα-treated and isotype control at day 4 p.i. (n = 5 ± SEM, * <i>p</i> < 0.05 based on Mann-Whitney test with respect to isotype control). (E) Histological analysis of the liver of anti-TNFα-treated mice, isotype antibody-treated mice harvested at day 4 p.i., and uninfected mice as indicated (n = 3 per group). Images were captured at 20x and 40x magnification. Representative sections from two independent experiments are shown (scale bar– 100μm).</p

Survival rate, clinical score, viremia and organ viral titers in DENV2-infected mice born to DENV1-immune or naïve mothers.

<p>5-6-weeks old A129 mice born to either DENV1-immune (black squares/bars) or naïve mothers (open square/bar) were iv infected with 10⁶ PFU of D2Y98P-PP1, a DENV2 strain. (A) Survival rate (n = 7–10). (B) Mean clinical score. 0: Healthy; 1: Ruffled Fur; 2: Hunched back; 3: Severe Diarrhea; 4: Lethargic; 5: Moribund. (C) Viremia. The number of infectious viral particles in serum was determined by plaque assay at day 2 and 4 p.i. (n = 5 mice per group per time point ± SEM). Dotted line denotes detection limit. (D) Viral load in brain, liver, spleen, brachial/ axillary lymph nodes, and intestines at day 4 p.i. was measured by plaque assay (n = 5 ± SEM). (E) Vascular leakage was assessed at day 4 p.i. using Evans’s blue dye. Five mice per group were iv injected with the dye and euthanized 2 hours later. After perfusion with PBS, various organs were harvested and processed for Evan’s blue dye extraction and quantification. Results are expressed as OD absorbance per gram of wet tissue (± SEM) and compared to uninfected control animals. Legend: DENV2-infected mice born to dengue naive mothers (open bar); DENV2-infected mice born to DENV1-immune mothers (black bar) and uninfected mice (striped bar). * <i>p</i><0.05 based on 1-way ANOVA with Bonferroni’s post-test for 1C and 2-way ANOVA with Bonferroni’s post-test for 1D and E. For each panel, one representative set of at least three independent experiments is shown.</p

DENV-specific antibody titers in DENV1-immune mothers and their offspring.

<p>Five to six weeks old naïve female A129 mice were infected iv with 10⁶ PFU of DENV1 and bled at 6 weeks post-infection (n = 3–5) (A&B). A129 mice born to naïve or DENV1-immune mothers were bled at the age of 5–6 weeks old (n = 5) (C&D). The antibody titers were determined by indirect ELISA against UV-inactivated DENV1/DENV2 particles as indicated. (A, C) Total IgG titers against DENV1 or DENV2 as indicated on the top of the graphs. (B, D) Anti-DENV1 IgG1, IgG2a and IgG2b titers. Titers were determined as the lowest dilution factor that gives an OD value of 3 times the mean blank. Dotted line denotes the lowest dilution of the sera and a nominal titer of 50 or 66.7 was assigned to samples with titers below the lowest dilution.</p

Enhancing properties of sera from 5–6 weeks old mice born to DENV1-immune mothers.

<p>K562 cells were infected with a mixture of DENV2-D2Y98P-PP1 and serially diluted pooled sera from mice born to DENV1-immune mothers (circle) or that from naïve mothers (square). Virus titers in supernatant were determined by plaque assay in BHK cells and expressed as fold enhancement (with respect to the average plaques obtained in the virus alone control).</p

Systemic levels of differentially modulated cytokines and chemokines in DENV2-infected A129 mice born to DENV1-immune or naïve mothers at day 4 p.i.

<p>Five to six weeks old A129 mice born to either DENV1-immune or naïve mothers were iv infected with 10⁶ PFU of D2Y98P-PP1. At day 2 or 4 p.i., the mice were terminally bled and the systemic levels of cytokines were determined by multiplex ELISA. Average levels of the differentially modulated cytokines (n = 5 each for 2–3 independent experiments) in the sera of DENV2-infected mice born to DENV1-immune mothers (black bars), DENV2-infected mice born to naïve mothers (open bars) and age-matched uninfected mice (stripe bars) were shown and error bar denotes standard deviation between experiments. Fold change were obtained by taking the average cytokine levels of the DENV2-infected mice born to the naïve mothers as reference. Legend: ^# Fold change is estimated as levels in DENV2-infected mice born to DENV1-immune mothers were above upper detection limit of the multiplex assay. *Levels in DENV2-infected mice born to DENV1-immune mothers were not significantly different from the basal levels in the uninfected control mice.</p

DENV antigen detection in the liver from DENV- infected A129 mice born to DENV1-immune or naïve mothers.

<p>Five to six weeks old A129 mice born to either DENV1-immune or naïve mothers were iv infected with 10⁶ PFU of D2Y98P-PP1. At day 4 p.i., tissues were harvested, frozen-sectioned, and stained for DENV NS3 (red) and cellular markers against endothelial cells (CD31, green), and monocytes (cd11b, blue). Representative sections from 2 independent experiments, each with n = 3 animals are shown (scale bar– 10μm). Individual channels corresponding to the merged images and co-localization images are presented.</p