Antibody-Dependent Enhancement of Dengue Virus Infection in Primary Human Macrophages; Balancing Higher Fusion against Antiviral Responses

Abstract

The dogma is that the human immune system protects us against pathogens. Yet, several viruses, like dengue virus, antagonize the hosts' antibodies to enhance their viral load and disease severity; a phenomenon called antibody-dependent enhancement of infection. This study offers novel insights in the molecular mechanism of antibody-mediated enhancement (ADE) of dengue virus infection in primary human macrophages. No differences were observed in the number of bound and internalized DENV particles following infection in the absence and presence of enhancing concentrations of antibodies. Yet, we did find an increase in membrane fusion activity during ADE of DENV infection. The higher fusion activity is coupled to a low antiviral response early in infection and subsequently a higher infection efficiency. Apparently, subtle enhancements early in the viral life cycle cascades into strong effects on infection, virus production and immune response. Importantly, and in contrast to other studies, the antibody-opsonized virus particles do not trigger immune suppression and remain sensitive to interferon. Additionally, this study gives insight in how human macrophages interact and respond to viral infections and the tight regulation thereof under various conditions of infection.

Figure 1. Antibody-dependent enhancement of DENV2 infection of primary human macrophages is dose-dependent.

Macrophages were incubated with DENV2, strain 16681 at MOI 1 which had been pre-incubated for 1 h with increasing concentrations of monoclonal human antibody (#753 C6; light grey, or #751 A2; dark grey). At 48 hpi, virus production was determined by plaque assay on BHK-15 cells. Shown is the SEM of duplicates. The figure is representative for two independent experiments. These antibodies are described more in-depth in a previous publication.

Figure 2. Antibodies and infection at high MOI enhance virus translation.

Viral translation was determined per cell on the intracellular pool of envelope protein. Macrophages were infected with DENV2. At 24–26 h, cells were stained with the anti-envelope antibody 4G2 and analysed by flow cytometry. Mean fluorescence intensities were normalized to the sample with the highest intensity of the donor. Up to 11 blood donors were used and six donors were used twice. Statistical analysis was done by One way ANOVA with Bonferroni post-test correction; *(p ≤ 0.05), **(p ≤ 0.01) ***(p ≤ 0.0001), n.s.: non-significant.

Figure 3. Antibodies do not alter the efficiency of dengue virus to bind or enter into primary human macrophages, whilst promoting fusion within primary macrophages.

(A) DENV2 binding and uptake in primary macrophages was determined at 1 hpi by qRT-PCR using template-specific primers in combination with RNAse A treatment. Extracellular virus was removed by shaving the cells with a high-salt-high-pH buffer for 2 min. Squares show the total number of virus particles that had bound or entered cells. Circles depict entered viral genomes, while triangles show negative-sense RNA genomes. Shown are 4 donors with each condition in duplicate. (B) Macrophages were infected with PKH67-labelled DENV at MOI 1 (red), MOI 1-ADE (blue) or MOI 5 (green). At 1 h (filled) or 2 h (striped) of incubation, the cells were shaved and fixed prior to analysis by flow cytometry. Cell entry was normalized to MOI 1, and the mean fluorescence intensity (MFI) was normalized to the negative control. Shown are the SEM of 5 donors. At MOI 1, 13 ± 3% of the cells were positive for PKH67-labelled DENV entry. (B–E) The fusion activity of DENV2 within primary human macrophages was determined at 30 min by microscopy using the self-quenching fluorophore DiD. Pictures were taken randomly and analysed for fusion activity (C), and the fraction of fusion-positive cells (D). All values were normalized to MOI 1 of the same donor (C) or infection in absence of antibodies (D,E). At MOI 1, the average number of fusion-positive cells was 17 ± 2.2%. (E) An overview of connected values of fusion activity when primary human macrophages are infected in the presence of enhancing antibodies (ADE), and when infected in the presence of an isotype (IgG). The inset shows the correlation between enhancement of fusion activity at 30 min post infection and the subsequent enhancement in virus production at 26 hpi. All values are given as percentage of the results obtained at MOI 1. Shown are the normalized SEM of up to 12 donors (A–C) and 5 experiments (D,E). Statistical analysis was done by 2-tailed t-test; *(P ≤ 0.05), **(P ≤ 0.01), ***(P ≤ 0.0005).

Figure 4. Dengue virus infection of macrophages induces an antiviral response, which depends on the viral load.

(A,D) Primary human macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, and matched infection (MOI 5 or MOI 2½). Total RNA was isolated at 24 h (A) and 2 h (D). Gene expression was investigated by microarray. Genes whose average expression had an absolute fold change of at least 1.5-fold over the mock were selected. The Venn diagram shows how these genes are connected with the various infection conditions. (A) is based on 3 donors and (D) on 4 donors. (B,C,E) Gene ontology of DAVID pathway analysis was used to annotated genes at 24 h (B,C) and at 2 h (E). (B) shows the ontological analysis of the genes that were shared among the three conditions (see A, triangle, 100 genes).(C) shows the analysis of the ADE-effect (see A, 460 genes total; 318 + 142). (E) shows the ontological analysis of the genes that were shared among the three conditions at 2 h (see D, triangle, 160 genes). The x-axis shows the enrichment of the term within our selection relative to the DAVID database. The y-axis shows the significance calculated with 1-tailed Fisher Exact statistical analysis. Gene ontology terms with at least 5-fold enrichment and a p-value of ≤1 ∙ 10⁻⁴ are considered relevant.

Figure 5. Early type-I-interferon (IFN)-mediated responses significantly determine the outcome of infection.

(A) Primary macrophages were infected with DENV2 at MOI 1, MOI 1-ADE, MOI 5, and MOI 10. Culture supernatants were harvested at 24 h and UV-inactivated. Inactivated supernatants were used to pre-activate Vero cells prior to infection with VSV at MOI 0.1. Shown is the SEM of 3 donors, with infection performed in duplicate and duplicates of this assay. Statistical analysis was done with One way ANOVA with post-test Bonferroni compensation; **(p ≤ 0.01) and n.s.; non-significant. (B) IFNα protects primary human macrophages against DENV2 infection when applied during the early (0–2 hpi) time points, and not during late (8 hpi) time points. Macrophages were infected with DENV2 at MOI 1, MOI 1-ADE and MOI 5. At the designated time points, 1 IU of recombinant human IFNα2a was added to the culture. At 26 hpi, the viral titre in the supernatant was determined by qRT-PCR and normalized relative to the unperturbed condition (No IFNα). Shown is the SEM of 4–5 donors, each condition in duplicate. (C) Type I IFN signalling was blocked by pre-incubating macrophages for 2 h with an antibody against the IFNαβR. After incubation, cells were infected at MOI 1, MOI 1-ADE and MOI 5. Supernatants were sampled at 26 hpi and virus production was determined by qRT-PCR. Addition of 10 units of IFNα at 0 hpi served as a control for IFNαβR-blocking. Shown is the normalized SEM of 2 donors, each condition in duplicate.

Figure 6. Molecular mechanisms involved in antibody-dependent enhancement of dengue virus infection in primary human macrophages.

Antibody-dependent infection does not enhance binding or entry of the virus to the cells. Yet, the membrane fusion potential within the endosomes of the macrophage is increased. Thanks to the unaltered characteristics of binding and entry, ADE does not trigger endogenous interferon-responses which thus allows the virus to replicate freely during the early stages of infection. ADE can be mimicked in terms of the number of infected cells and burst size by infection at high MOI in absence of antibodies. Yet high MOI leads to more binding, entry, fusion, and as a consequence induction of an IFN response. The presence of an early IFN response significantly reduces virus replication and production. ADE is thus based on higher fusion but due to the absence of an early IFN response, it remains unnoticed by the cell allowing virus replication to higher titres.