Antibody (Ab)-dependent enhancement (ADE) is a hypothesized mechanism of increased disease severity during secondary dengue virus (DENV) infection. This study investigates Ab-dependent cell cytotoxicity (ADCC) in counteracting ADE. In our system, DENV and DENV-immune sera were added to peripheral blood mononuclear cells (PBMCs), and ADE and NK cell activation were simultaneously monitored. ADE was detected in monocytes and a concurrent activation of NK cells was observed. Activated NK cells expressed IFN-γ and CD107a. IFN-γ was detected at 24 hours (24 h) followed by a rapid decline; CD107a expression peaked at 48 h and persisted for >7 days. Optimal activation of NK cells required the presence of enhancement serum together with ADE-affected monocytes and soluble factors, suggesting the coexistence of the counteractive ADCC Abs, in the same ADE-serum, capable of strongly promoting NK cell activation. The function of NK cells against ADE was demonstrated using a depletion assay. NK cell-depleted PBMCs had increased ADE as compared to whole PBMCs. Conversely, adding activated NK cells back into the NK-depleted-PBMCs or to purified monocytes decreased ADE. Blocking IFN-γ expression also increased ADE. The study suggests that under ADE conditions, NK cells can be activated by ADCC Abs and can control the magnitude of ADE.

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This study describes an antibody-dependent NK cell degranulation assay, as a biomarker to assess antibody-dependent cellular cytotoxicity (ADCC) response in influenza plasma and for antibody therapies against influenza infection. The concentration of neutralizing antibodies (NAbs) against the hemagglutinin receptor of influenza viruses is a current determinant in protection against infection, particularly following receipt of the seasonal influenza vaccine. However, this is a limited assessment of protection, because: (i) NAb titers that incur full protection vary; and (ii) NAb titers do not account for the entire breadth of antibody responses against viral infection. Previous reports have indicated that antibodies that prime ADCC play a vital role in controlling influenza infections, and thus should be quantified for assessing protection against influenza. This report demonstrates a non-radioactive assay that assesses NK cell activation as a marker of ADCC, in which NK cells interact with opsonized viral antigen expressed on the surface of infected Raji target cells resulting in effector cell degranulation (surrogate CD107a expression). A positive correlation was determined between HAI titers and sustained NK cell activation, although NK cell activation was seen in plasma samples with HAI titers below 40 and varied amongst samples with high HAI titers. Furthermore, sustained NK cell degranulation was determined for influenza-vaccinated transchromosomic bovine intravenous immunoglobulin, indicating the potential utility of this therapy for influenza treatment. We conclude that this assay is reproducible and relevant.

Background The dengue virus (DENV) group is composed of 4 serotypes, DENV-1, -2, -3 and -4. The 4 serotypes of DENV share70% sequence homology, therefore cross-reactivity among 4 serotypes is common. Cross-reactive antibodies (Ab) can cause Ab-dependent enhancement (ADE) of viral infection. ADE is thought to be an underlying mechanism of elevated immune-pathology associated with severe dengue diseases. Human monocytes and macrophages are target cells for ADE. This study investigates NK cell-mediated Ab-dependent cell cytotoxicity (ADCC) as a critical control mechanism following an established ADE intracellularly. Methods To model the physiological situation, whole human PBMCs were infected with DENV in the presence of autologous serially diluted sera. ADE in monocytes was detected by intracellular staining of viral antigens and by RT-PCR. NK cell activation was monitored in the same cultures by a degranulation marker CD107a. Other NK cell markers, cell proliferation and cytokine/chemokines were investigated. Results ADE was only detected in monocytes with the serially diluted immune sera. NK cell activation occurred concomitantly with the detection of ADE. The peak of the ADCC response was 48 hours post infection, but the response was still detectable at day 7. Minimal activation of T cells was observed. The power of ADE in monocytes ranged between 3 to 10.2 folds when NK cells were depleted as compared to 0.6 to 1.8 folds under normal condition. Activated NK cells had elevated expression of CD69, CD335, CD336, and CD96, but had downregulated CD337 and CD314. Conclusion ADCC is activated when DENV escapes from Ab neutralization and establishes infection in host cells, and it can act as the first line of defense to combat ADE.

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