Antibody-mediated enhancement aggravates chikungunya virus infection and disease severity

Abstract

The arthropod-transmitted chikungunya virus (CHIKV) causes a flu-like disease that is characterized by incapacitating arthralgia. The re-emergence of CHIKV and the continual risk of new epidemics have reignited research in CHIKV pathogenesis. Virus-specific antibodies have been shown to control virus clearance, but antibodies present at sub-neutralizing concentrations can also augment virus infection that exacerbates disease severity. To explore this occurrence, CHIKV infection was investigated in the presence of CHIKV-specific antibodies in both primary human cells and a murine macrophage cell line, RAW264.7. Enhanced attachment of CHIKV to the primary human monocytes and B cells was observed while increased viral replication was detected in RAW264.7 cells. Blocking of specific Fc receptors (FcγRs) led to the abrogation of these observations. Furthermore, experimental infection in adult mice showed that animals had higher viral RNA loads and endured more severe joint inflammation in the presence of sub-neutralizing concentrations of CHIKV-specific antibodies. In addition, CHIKV infection in 11 days old mice under enhancing condition resulted in higher muscles viral RNA load detected and death. These observations provide the first evidence of antibody-mediated enhancement in CHIKV infection and pathogenesis and could also be relevant for other important arboviruses such as Zika virus.

Figure 1

Enhancement of CHIKV infection in purified primary human cells. Primary human (a,c) monocytes and (b,d) B cells (2 × 10⁶ cells per infection) were infected with CHIKV (moi 10) in the presence (enhanced) or absence (non-enhanced) of diluted CHIKV-specific patient plasma containing total IgG at a concentration of 3.6 ± 2.9 μg/ml. (a,b) Detection of CHIKV antigen and (c,d) viral RNA load were determined at 0 and 6 hpi. Level of CHIKV antigen detected is expressed as fold enhancement relative to the non-enhanced infection controls. Data shown are mean ± SD from 3 independent experiments by parametric unpaired t test (**P = 0.0015 for 0 hpi monocytes; *P = 0.0458 for 0 hpi B cells and **P = 0.0053 for 6 hpi B cells). Expression of FcγRs in (e) monocytes and (f) B cells. Primary human (g) monocytes and (h) B cells (2 × 10⁶ cells per infection) were treated with 10 μg of FcγRs blocking agent prior to CHIKV infection (moi 10) under enhancing conditions (as above). Percentage of infection is expressed as level of CHIKV antigen detected relative to the non-treated infection controls at 0 hpi. Data are presented as mean ± SD from 4 independent experiments by Mann-Whitney U test (*P = 0.0286 for monocytes and *P = 0.0143 for B cells).

Figure 2

Enhancement of CHIKV infection is mediated via Fcγ receptors. (a) Expression of FcγRI, FcγRII and FcγRIII was determined in FcγR-expressing ST486 cell lines by flow cytometry (n = 3). CHIKV (moi 10) was used to infect (b) non-FcγR-expressing, (c) FcγRI-expressing, (d) FcγRII-expressing and (e) FcγRIII-expressing ST486 cell lines (2 × 10⁶ cells per infection) in the presence (enhanced) or absence (non-enhanced) of diluted patient plasma containing total IgG at a concentration of 3.6 ± 2.9 μg/ml (n = 9). Level of CHIKV antigen was determined at 0 and 6 hpi. Level of CHIKV antigen detected is expressed as fold enhancement relative to the non-enhanced infection controls. Data are presented as mean ± SD by Mann-Whitney U test (***P < 0.0001 for level of CHIKV infection observed at both 0 and 6 hpi in FcγRI and FcγRII expressing cells).

Figure 3

CHIKV infection enhancement in macrophages from humans and mice. (a) Expression of FcγRs in human monocytes-derived macrophages (MDMs) was detected with flow cytometry. Human MDMs (2 × 10⁶ cells per infection) were infected with Zs-Green tagged CHIKV (moi 10) either in the presence (enhanced) or absence (non-enhanced) of diluted CHIKV patient plasma containing total IgG at a concentration of 1.8 ± 1.45 μg/ml. (b) Level of infection and (c) viral RNA load were determined at 24 and 48 hpi. Level of infection was determined by the amount of Zs-Green positive cells and is expressed as fold enhancement relative to the non-enhanced infection controls. Data shown are mean ± SD from 4 independent experiments by Mann-Whitney U test (*P = 0.0317 for level of infection at 24 hpi). (d) FcγRs expression in RAW264.7 cells was determined by flow cytometry. CHIKV (moi 10) was used to infect RAW264.7 cells (2 × 10⁶ cells per infection) in the presence (enhanced) or absence (non-enhanced) of diluted CHIKV-specific mice sera at concentration of ~2 µg/ml. (e) Detection of CHIKV antigen and (f) viral RNA load were determined in these infected cells at 0, 6 and 12 hpi. Level of CHIKV antigen detected is expressed as fold enhancement relative to non-enhanced infection controls. Data shown are mean ± SD from 3 independent experiments by parametric unpaired t test (*P = 0.0341 and 0.0350 for 0 and 6 hpi respectively for level of infection in RAW264.7 cells; ***P = 0.0006 for 6 hpi and *P = 0.0269 for 12 hpi for viral RNA load in infected RAW264.7 cells).

Figure 4

Levels of Type I IFN and pro-inflammatory genes upon CHIKV infection under enhancing conditions. RAW264.7 cells (2 × 10⁶ cells per infection) were infected with CHIKV (moi 10) under both enhancing and non-enhancing conditions and infected cells were harvested at 0, 6 and 12 hpi for gene expression study on selected pro-inflammatory and Type I IFN related genes by qRT-PCR. (a) Signature of gene expression across all stipulated time points displayed as a two-way clustered heat plot. Data are normalized to mock-infected controls. (b) Elevated expression of key mediators during enhanced CHIKV infection at 12 hpi. Data are normalized relative to mock-infected controls. Data are presented as mean ± SD from 3 independent experiments by parametric unpaired t test (*P = 0.0299 for IL-6; *P = 0.0117 for iNOS; *P = 0.0184 for IFNα; *P = 0.0498 for IRF3; *P = 0.0259 for IRF9; *P < 0.0001 for Viperin; *P = 0.0466 for ISG15).

Figure 5

Exacerbation of joint inflammation in adult mice. (a,b) Three-weeks old, C57BL/6 WT female mice (n = 5 animals per group) were infected via footpad inoculation with 10⁶ PFU of CHIKV. Immediately, these mice were administered intraperitoneally with ~2 µg/ml (total IgG concentration) mice sera from CHIKV-infected mice (enhanced) or PBS (non-enhanced). Daily assessment of (a) viremia and (b) disease score were performed. All Data are presented as mean ± SD by Mann-Whitney U test (*P = 0.0278, *P = 0.0159, *P = 0.0159 and *P = 0.0297 for 2, 3, 4 and 5 dpi viremia respectively; **P = 0.0040, **P = 0.0079, **P = 0.0060, **P = 0.0079, **P = 0.0079, *P = 0.0104, *P = 0.0172, **P = 0.0040, **P = 0.0060, *P = 0.0106 and *P = 0.0106 for 3, 4, 5, 6, 7, 8, 9, 10, 11, 13 and 14 dpi disease score respectively). Immune-phenotyping was performed for (c) footpad and (d) popliteal lymph node on 6 dpi. Results are displayed as number of neutrophils, CD4+ T cells and monocytes per organ from 4 to 10 animals per group. (e) Total RNA was extracted from the joint footpads of infected mice (n = 5 to 10 animals per group) and qRT-PCR was performed to detect for the expression of crucial immune genes. Gene expression data are expressed as fold expression relative to the mock-infected mice. Mice sacrificed for immune-phenotyping and gene expression studies were infected as described for (a,b). All Data are presented as mean ± SD by Mann-Whitney U test (*P = 0.0186, *P = 0.0326 and *P = 0.0185 for neutrophils, monocytes and CD4+ T cells infiltration into respective organs; *P = 0.0276, *P = 0.0376 and *P = 0.0376 for IFNγ, DEF14 and IL-10 expression respectively).

Figure 6

Aggravated disease outcome in CHIKV-infected IFNαR+/− mice. Three-weeks old IFNαR+/− female mice (n = 7 to 11 animals per group) were infected via footpad inoculation with 10⁶ PFU of CHIKV. Immediately, these mice were administered intraperitoneally with ~2 µg/ml (total IgG concentration) mice sera from CHIKV-infected mice (enhanced) or PBS (non-enhanced). Daily assessment of (a) viremia and (b) disease score were performed. Data are presented as mean ± SD by Mann-Whitney U test (**P = 0.0027, **P = 0.0011, ***P = 0.0009, ***P = 0.0005, **P = 0.0017 and *P = 0.0159 for 2, 3, 4, 5, 6 and 7 dpi viremia respectively; ⁺P = 0.0125, ⁺⁺⁺P < 0.0001, ⁺⁺P = 0.0015 and ⁺P = 0.0277 for 4, 5, 6 and 7 dpi viremia respectively; *P = 0.0109 and *P = 0.0306 for 10 and 11 dpi disease score respectively; ⁺⁺⁺P < 0.0001, ⁺⁺⁺P = 0.0006, ⁺P = 0.0109, ⁺P = 0.0277, ⁺P = 0.0031, ⁺P = 0.0095 and ⁺P = 0.0031 for 1, 6, 7, 8, 9, 10 and 11 dpi disease score respectively. (*) indicates significance comparing between CHIKV-infected IFNαR+/− (blue) and WT animals (black); (+) indicates significance comparing between IFNαR+/− mice infected under enhanced (red) and non-enhanced (blue) conditions.

Figure 7

Enhanced infection-associated lethality in young mice. (a) 11-day old C57BL/6 (female, n = 10 animals per group) were inoculated intradermally with 10⁶ PFU of CHIKV-21. Immediately after, these mice were given intraperitoneally either human naive polyvalent antibodies or purified CHIKVIGs at different concentrations. Survival of these animals was monitored over a period of 3 weeks. (b,c) 11 days old C57BL/6 (female, n = 3 animals per group) were infected as described in (a) but were intraperitoneally given purified human CHIKVIGs at 10 µg/kg. Control animals were given 10 µg/kg of human naive polyvalent antibodies. Viral RNA load in the serum, liver and muscle were determined at both (b) 3 and (c) 6 dpi. All data are presented as mean ± SD. *p < 0.05 by parametric unpaired t test.

Figure 8

Impact of antibody-mediated enhancement in CHIKV infection. Virus-antibody complexes are formed when antibodies are present in sub-neutralizing concentrations and are taken up by cells expressing FcγRs via either extrinsic or intrinsic pathways. Extrinsic pathways result in increased virus attachment and possibly entry with no visible benefits to virus replication. Whereas in intrinsic pathways, it can lead to enhanced anti-inflammatory or enhanced pro-inflammatory infection. Enhanced anti-inflammatory infection downplays the inflammatory response associated with normal infection and instead upregulates the anti-inflammatory response leading to heightened infection and viral replication. In enhanced pro-inflammatory infection, it leads to an increased in active infection, without negative suppression of pro-inflammatory immune response.