Guillain-Barre syndrome after SARS-CoV-2 infection in an international prospective cohort study

In the wake of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, an increasing number of patients with neurological disorders, including Guillain-Barre syndrome (GBS), have been reported following this infection. It remains unclear, however, if these cases are coincidental or not, as most publications were case reports or small regional retrospective cohort studies. The International GBS Outcome Study is an ongoing prospective observational cohort study enrolling patients with GBS within 2 weeks from onset of weakness. Data from patients included in this study, between 30 January 2020 and 30 May 2020, were used to investigate clinical and laboratory signs of a preceding or concurrent SARS-CoV-2 infection and to describe the associated clinical phenotype and disease course. Patients were classified according to the SARS-CoV-2 case definitions of the European Centre for Disease Prevention and Control and laboratory recommendations of the World Health Organization. Forty-nine patients with GBS were included, of whom eight (16%) had a confirmed and three (6%) a probable SARS-CoV-2 infection. Nine of these 11 patients had no serological evidence of other recent preceding infections associated with GBS, whereas two had serological evidence of a recent Campylobacter jejuni infection. Patients with a confirmed or probable SARS-CoV-2 infection frequently had a sensorimotor variant 8/11 (73%) and facial palsy 7/11 (64%). The eight patients who underwent electrophysiological examination all had a demyelinating subtype, which was more prevalent than the other patients included in the same time window [14/30 (47%), P = 0.012] as well as historical region and age-matched control subjects included in the International GBS Outcome Study before the pandemic [23/44 (52%), P = 0.016]. The median time from the onset of infection to neurological symptoms was 16 days (interquartile range 12-22). Patients with SARS-CoV-2 infection shared uniform neurological features, similar to those previously described in other post-viral GBS patients. The frequency (22%) of a preceding SARS-CoV-2 infection in our study population was higher than estimates of the contemporaneous background prevalence of SARS-CoV-2, which may be a result of recruitment bias during the pandemic, but could also indicate that GBS may rarely follow a recent SARS-CoV-2 infection. Consistent with previous studies, we found no increase in patient recruitment during the pandemic for our ongoing International GBS Outcome Study compared to previous years, making a strong relationship of GBS with SARS-CoV-2 unlikely. A case-control study is required to determine if there is a causative link or not

Second intravenous immunoglobulin dose in patients with Guillain-Barre syndrome with poor prognosis (SID-GBS):a double-blind, randomised, placebo-controlled trial

Background Treatment with one standard dose (2 g/kg) of intravenous immunoglobulin is insufficient in a proportion of patients with severe Guillain-Barre syndrome. Worldwide, around 25% of patients severely affected with the syndrome are given a second intravenous immunoglobulin dose (SID), although it has not been proven effective. We aimed to investigate whether a SID is effective in patients with Guillain-Barre syndrome with a predicted poor outcome. Methods In this randomised, double-blind, placebo-controlled trial (SID-GBS), we included patients (>= 12 years) with Guillain-Barre syndrome admitted to one of 59 participating hospitals in the Netherlands. Patients were included on the first day of standard intravenous immunoglobulin treatment (2 g/kg over 5 days). Only patients with a poor prognosis (score of >= 6) according to the modified Erasmus Guillain-Barre syndrome Outcome Score were randomly assigned, via block randomisation stratified by centre, to SID (2 g/kg over 5 days) or to placebo, 7-9 days after inclusion. Patients, outcome adjudicators, monitors, and the steering committee were masked to treatment allocation. The primary outcome measure was the Guillain-Barre syndrome disability score 4 weeks after inclusion. All patients in whom allocated trial medication was started were included in the modified intention-to-treat analysis. Findings Between Feb 16, 2010, and June 5, 2018, 327 of 339 patients assessed for eligibility were included. 112 had a poor prognosis. Of those, 93 patients with a poor prognosis were included in the modified intention-to-treat analysis: 49 (53%) received SID and 44 (47%) received placebo. The adjusted common odds ratio for improvement on the Guillain-Barre syndrome disability score at 4 weeks was 1.4 (95% CI 0.6-3.3; p=0.45). Patients given SID had more serious adverse events (35% vs 16% in the first 30 days), including thromboembolic events, than those in the placebo group. Four patients died in the intervention group (13-24 weeks after randomisation). Interpretation Our study does not provide evidence that patients with Guillain-Barre syndrome with a poor prognosis benefit from a second intravenous immunoglobulin course; moreover, it entails a risk of serious adverse events. Therefore, a second intravenous immunoglobulin course should not be considered for treatment of Guillain-Barre syndrome because of a poor prognosis. The results indicate the need for treatment trials with other immune modulators in patients severely affected by Guillain-Barre syndrome. Funding Prinses Beatrix Spierfonds and Sanquin Plasma Products. Copyright (C) 2021 Elsevier Ltd. All rights reserved

Respiratory Syncytial Virus Induced Type I IFN Production by pDC Is Regulated by RSV-Infected Airway Epithelial Cells, RSV-Exposed Monocytes and Virus Specific Antibodies

<div><p>Innate immune responses elicited upon virus exposure are crucial for the effective eradication of viruses, the onset of adaptive immune responses and for establishing proper immune memory. Respiratory syncytial virus (RSV) is responsible for a high disease burden in neonates and immune compromised individuals, causing severe lower respiratory tract infections. During primary infections exuberant innate immune responses may contribute to disease severity. Furthermore, immune memory is often insufficient to protect during RSV re-exposure, which results in frequent symptomatic reinfections. Therefore, identifying the cell types and pattern recognition receptors (PRRs) involved in RSV-specific innate immune responses is necessary to understand incomplete immunity against RSV. We investigated the innate cellular response triggered upon infection of epithelial cells and peripheral blood mononuclear cells. We show that CD14⁺ myeloid cells and epithelial cells are the major source of IL-8 and inflammatory cytokines, IL-6 and TNF-α, when exposed to live RSV Three routes of RSV-induced IFN-α production can be distinguished that depend on the cross-talk of different cell types and the presence or absence of virus specific antibodies, whereby pDC are the ultimate source of IFN-α. RSV-specific antibodies facilitate direct TLR7 access into endosomal compartments, while in the absence of antibodies, infection of monocytes or epithelial cells is necessary to provide an early source of type I interferons, required to engage the IFN-α,β receptor (IFNAR)-mediated pathway of IFN-α production by pDC. However, at high pDC density infection with RSV causes IFN-α production without the need for a second party cell. Our study shows that cellular context and immune status are factors affecting innate immune responses to RSV. These issues should therefore be addressed during the process of vaccine development and other interventions for RSV disease.</p> </div

Virus specific polyclonal antibodies in human serum increase RSV binding to monocytes and B cells.

<p>(<b>A</b>) The effect of autologous serum, IgG-depleted serum and palivizumab in FCS on binding of RSV to monocytes, B cells and NK cells was measured after 1 hour incubation at 4°C. (<b>B</b>) Polyclonal antibodies (IVIG) increase binding of RSV to CD14^+/CD16^neg. cells. Data shown represent the mean ± SEM of triplicate measurements in two different donors (for A and B) and were analyzed using the Kruskal-Wallis test followed by Dunn's Multiple Comparison analysis, *P< 0.05, **P <0.01. Experiments were performed in 3 additional donors with similar results.</p

RSV-specific antibodies inhibit RSV-induced IFN-α production in PBMC, but enhance IFN-α production in CD14⁺ cell depleted PBMC.

<p>(<b>A</b>) Inhibition of RSV infection in pDC. Lineage^neg., MHC-II^high, BDCA-4⁺ pDC are partially infected with rgRSV224 after a period of 20 hours. Infection is blocked after UV inactivation, after neutralization in 10% fresh human serum or in 5µg/ml Palivizumab. Similar results were obtained with sera from all donors used during our studies. IFN-α in supernatant of RSV-A2 exposed PBMC-CD14⁺ cell cultures (<b>B</b>) and rgRSV224 exposed PBMC-CD14⁺ cells or PBMC, (<b>C</b>) was measured after 20 hrs. The role of virus specific antibodies on the cytokine response was tested by removing IgGs from AS with protein G Sepharose^® beads and reconstitution with 2 mg/ml IVIG (<b>B</b>) or 5 µg/ml palivizumab (<b>B</b>, <b>C</b>). Experiments represent the mean ± SEM of experiments performed in 4 different donors and were analyzed using one way ANOVA followed by a Bonferroni post-test, **P <0.01.</p

CD14⁺ monocytes inhibit IFN-α production triggered by Ab-RSV via TLR7 in pDC.

<p>(<b>A</b>) IFN-α production in CD14⁺ cell depleted PBMCs induced by Ab-RSV complexes, TLR9 ligand ODN 2216 and TLR7 ligand imiquimod is decreased by blocking endosomal acidification with 50nM Bafilomycin A₁. (<b>B</b>) Ab-RSV-induced IFN-α production in CD14⁺ cell depleted PBMCs was abrogated in the presence of immune regulatory sequence (IRS) 661 (1.4 µM) a specific blocking agent for endosomal TLR7 and not by a scrambled control nucleotide. (<b>C</b>) pDC are the source of Ab-RSV induced, TLR7 mediated production of IFN-α, as shown by intracellular staining for IFN-α in Lineage (CD3^neg., CD14^neg., CD19^neg., CD16^neg., CD56^neg., Lin-1), MHC-II^high, BDCA-4⁺ cells. The inhibitor brefeldin A was added at different time points post infection (the time points when BFA was added are given on the X-axis). Cytokines were allowed to accumulate for 10 hrs. after addition of BFA. (<b>D</b>) Purified pDC (obtained by negative selection removing CD3⁺, CD19⁺ and CD16⁺ cells from fresh PBMC, followed by FACS purification of the BDCA-4⁺ cell population, which resulted in > 95% pure pDC) produce IFN-α upon infection with RSV. This response is abrogated after UV inactivation of RSV. In AS, both live RSV and UV-inactivated RSV induced IFN-α production to a similar extent. One representative experiment out of two performed with pDC isolated from two different donors is shown. (<b>E</b>) IFN-α production by Ab-RSV in purified pDC is blocked by IRS661 (1.4µM). (<b>F</b>) TLR1,-2 (PAM3CSK4, Peptidoglycan) and TLR4 (LPS) ligands suppress TLR9-triggered (ODN 2216) IFN-α production, but do not affect TLR7 (Gardiquimod) induced IFN-α production. All data represent mean ± SEM of triplicate measurements within 1 donor and analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P< 0.05, **P <0.01. Experiments were performed in 3 different donors with similar results.</p

IFN-α production induced by live RSV in PBMC depends on IFNAR signalling.

<p>PBMC were exposed to live RSV in the presence of interferon-α/β receptor (IFNAR) blocking antibody (5µg/ml), isotype control Ab or no Ab. Levels of IFN-α were determined via intracellular staining (<b>A</b>) or in 20hrs. supernatant by ELISA (<b>B</b>). IFN-α was trapped intracellular by BFA treatment initiated 6 hrs. after RSV infection, or at t=0 for the ODN 2216 control stimulus, because of different kinetics of anti-viral and ODN elicited IFN-α response. For both stimuli, intracellular staining for IFN-α was performed in CD3^neg., CD16^neg., CD19^neg., CD56^neg., MHCII⁺, BDCA-4⁺ cells after 10 hrs. BFA treatment. Experiments were performed in 3 different donors with similar results. Data represent the mean ± SEM of triplicate measurements within 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, **P <0.01.</p

IFN-α production induced by live RSV in pDC depends on CD14.

<p>The role of the TLR4/CD14/MD2 complex in the IFN-α and IL-6 response by PBMC after RSV infection (strain A2, MOI 5) was investigated (<b>A</b>) with a blocking monoclonal antibody specific for human TLR4 (20µg/ml), (<b>B</b>) the MD2 antagonist lipopolysaccharide from the bacterium <i>Rhodobacter </i><i>sphaeroides</i> (1µg/ml) or (<b>C</b>) via neutralization of CD14 with monoclonal antibody MY4 (10µg/ml). (<b>A</b>-<b>C</b>) Cytokine responses were measured by ELISA. Control stimuli; LPS: 10ng/ml, ODN: 5µg/ml. (<b>D</b>) A549 epithelial cells were co-cultured with PBMC, CD14⁺ cell depleted PBMC, pDC depleted PBMC, or CD14⁺ and BDCA-4⁺ double depleted PBMC, in the presence of RSV at MOI 5, ODN 2216 or no stimulus, for a period of 20 hrs. IL-6 and IFN-α production were measured by ELISA. Experiments were performed in 3 different donors with similar results. Data represent the mean ± SEM of triplicate measurements within 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test. ns not significant, *P<0.05, **P<0.01. </p

CD14⁺ cells are needed for the inflammatory cytokine response against RSV in PBMC cultures.

<p>(<b>A</b>) Confirmation of specific depletion of CD14⁺ and BDCA-4⁺ cells by FACS. M: monocyte gate, L: lymphocyte gate. Depletion of CD14⁺ cells leaves B cells (in M+L, CD19⁺ third row) and CD16⁺ monocyte/DC (in M, CD14^neg., CD16⁺, 2^nd row), cDC (in M+L, lineage^neg., MHCII⁺, CD11c⁺, 4^rd row) and pDC (in M+L, lineage^neg., MHCII⁺, BDCA-4⁺, 4^rd row) compartments intact. Depletion of BDCA-4⁺ cells removes pDC but does not affect CD14⁺CD16^neg., CD14⁺CD16⁺, CD14^neg.CD16⁺ cells in the M gate (2^nd row), nor B cells (3^rd row) and cDC (4^rd row). The contribution of single cell types to anti-RSV cytokine responses in PBMC cultures was evaluated by depletion of specific cell populations. Cytokines in supernatant from the remaining cell populations were measured after 20 hrs. exposure to RSV or UV-RSV in the presence or absence of autologous serum. (<b>B</b>) Depletion of CD14⁺ cells and BDCA-4⁺ cells was confirmed by stimulation of depleted cell populations by TLR ligands, ultra-pure LPS to confirm the absence of TLR4⁺ (monocytes) and ODN 2216 to confirm the absence of TLR9⁺ (pDC). (<b>C</b>) PBMC, CD14 monocyte-depleted PBMC, or pDC-depleted PBMC were cultured with live and UV inactivated RSV (A2, MOI 5) in the presence or absence of 10% autologous serum. Data represent the mean ± SEM of 3 measurements in 1 donor and were analyzed using one way ANOVA followed by a Bonferroni post-test, *P< 0.05, **P <0.01. Experiments were performed in 3 different donors with similar results.</p