Early autoimmunity and outcome in virus encephalitis: a retrospective study based on tissue-based assay

Abstract

To explore the autoimmune response and outcome in the central nervous system (CNS) at the onset of viral infection and correlation between autoantibodies and viruses.

Methods: A retrospective observational study was conducted in 121 patients (2016-2021) with a CNS viral infection confirmed via cerebrospinal fluid (CSF) next-generation sequencing (cohort A). Their clinical information was analysed and CSF samples were screened for autoantibodies against monkey cerebellum by tissue-based assay. In situ hybridisation was used to detect Epstein-Barr virus (EBV) in brain tissue of 8 patients with glial fibrillar acidic protein (GFAP)-IgG and nasopharyngeal carcinoma tissue of 2 patients with GFAP-IgG as control (cohort B).

Results: Among cohort A (male:female=79:42; median age: 42 (14-78) years old), 61 (50.4%) participants had detectable autoantibodies in CSF. Compared with other viruses, EBV increased the odds of having GFAP-IgG (OR 18.22, 95% CI 6.54 to 50.77, p<0.001). In cohort B, EBV was found in the brain tissue from two of eight (25.0%) patients with GFAP-IgG. Autoantibody-positive patients had a higher CSF protein level (median: 1126.00 (281.00-5352.00) vs 700.00 (76.70-2899.00), p<0.001), lower CSF chloride level (mean: 119.80±6.24 vs 122.84±5.26, p=0.005), lower ratios of CSF-glucose/serum-glucose (median: 0.50[0.13-0.94] vs 0.60[0.26-1.23], p=0.003), more meningitis (26/61 (42.6%) vs 12/60 (20.0%), p=0.007) and higher follow-up modified Rankin Scale scores (1 (0-6) vs 0 (0-3), p=0.037) compared with antibody-negative patients. A Kaplan-Meier analysis revealed that autoantibody-positive patients experienced significantly worse outcomes (p=0.031).

Conclusions: Autoimmune responses are found at the onset of viral encephalitis. EBV in the CNS increases the risk for autoimmunity to GFAP.

Keywords: autoimmune encephalitis; clinical neurology; neuroimmunology; neurovirology.

Figure 1

Indirect immunofluorescence patterns of autoantibodies against neurons, astrocytes or other antigens. (A) Typical immunofluorescence pattern of glial fibrillar acidic protein (GFAP)-IgG in the cerebellum, showing IgG bound to the astrocytes in all layers (arrows), which was confirmed by cell-based assay (CBA, figure a). (B) Immunofluorescence pattern of neuronal cellular surface-specific antibody overlapping N-methyl-D-aspartate receptor (NMDAR)-IgG (granular layer confirmed by CBA, figure b) with unknown antigen (molecular layer) in the cerebellum. (C) Immunofluorescence pattern of neuronal nucleus-specific antibody with unknown antigen. (D) Immunofluorescence pattern of Purkinje cell (PC)-specific antibody with unknown antigen. (E) Immunofluorescence pattern of overlapping GFAP-IgG and unknown neuron-specific antibody. (F) Immunofluorescence pattern of neuronal intermediate filament (NIF), confirmed as α-internexin (INA) by CBA (figure d). (G) Typical immunofluorescence pattern of antimyelin oligodendrocyte glycoprotein (MOG) IgG in the myelin of the cerebellum, which was confirmed by CBA (figure c).

Figure 2

Classification of virus and autoantibody. At the onset of herpes simplex virus (HSV) infection, 17/42 (10.5%) patients had antibodies against neuronal antigens (n=12, 28.6%), glial fibrillar acidic protein (GFAP) (n=3, 7.1%) or other antigens (n=2, 4.8%); at the onset of Epstein-Barr virus (EBV) infection, 24/30 (80.0%) patients had antibodies against neuronal antigens (n=3, 10.0%), GFAP (n=20, 66.7%) or other antigens (n=1, 3.3%); at the onset of varicella zoster virus (VZV), 19/47 (40.4%) patients had antibodies against neuronal antigens (n=12, 25.5%), GFAP (n=5, 10.6%) or other antigens (n=2, 4.3%). The one patient with cytomegalovirus had GFAP-IgG in the cerebrospinal fluid. The patients with EBV infection were more likely to have autoantibodies (24/30), compared with patients with HSV (17/42) or VZV (19/47) infection (p<0.001). More antibodies against neuronal antigens were detected in patients with HSV (12/17) or VZV (12/19) infection (EBV 3/24, p<0.001) while more antibodies against GFAP (20/24) were detected in patients with EBV infection (HSV 3/17, VZV 5/19, p<0.001). Ab, antibody.

Figure 3

Neurological status of the patients stratified by autoantibody presence in the CSF. The neurological status was measured using the mRS and was colour-coded. (A) Each patient’s mRS scores at admission, discharge and recent follow-up are shown in the chart. According to mRS scores at admission, there were no statistical differences in severity of infection between CSF-positive and CSF-negative patients. CSF-positive patients seemed to have worse neurological status at discharge, although there were no statistical differences. And CSF-positive patients had worse outcome according to the mRS scores in recent follow-up. In each group, there were no statistical difference in the mRS scores between patients with different virus infection, except that in the CSF-negative group, patients with HSV infection were worse than those with VZV infection at admission (median: 4 (2–5) vs 3 (0–5), p<0.001). (B, C, D) Bar charts of statistics on the mRS scores of patients with detectable anti-GFAP antibody (B, n=29, loss to follow-up rate (loss rate)=7/29), with other autoantibodies (C, n=32, loss rate=7/32) or without autoantibodies (D, n=60, loss rate=8/60). No significant differences were found among the loss to follow-up rates (loss rates) of the different groups (p=0.339). CSF-positive patients (n=45) with follow-up (median: 28 months; IQR: 16–42 months; range: 4–62 months). CSF-negative patients (n=52) with follow-up (median: 25 months; IQR: 15–44 months; range: 6–64 months). No significant differences were found in loss rates between CSF-positive and CSF-negative patients (p=0.075). (E) A Kaplan-Meier analysis with mRS ≥3 as the end point (log rank (Mantel-Cox), p=0.031). The CSF-positive patients were more likely to have poorer outcomes. There were no statistical differences in outcomes between patients with anti-GFAP antibodies and other antibodies. Ab, antibody; CSF, cerebrospinal fluid; EBV, Epstein-Barr virus; GFAP, glial fibrillar acidic protein; HSV, herpes simplex virus; mRS, modified Rankin Scale; VZV, varicella zoster virus.

Figure 4

Pathological evidence of EBV infection in the CNS of patients with GFAP-A. (A, B, C, D) Brain tissue was obtained from two previously reported patients with subacute encephalitis. (A, B, C) Tissue was obtained from patient no. 2 in the previous study. (D) Tissue was obtained from patient no. 6 in the previous study. (A, B) Inflammation was revealed in the meninges and brain parenchyma. Green arrow: astrocyte dystrophy. (C) Astrocytes with EBV infection, detected by ISH. Green solid arrow: EBV-infected astrocyte with degeneration. Green dashed arrow: EBV-infected astrocyte with normal morphology. (D) Neurons and astrocytes stained by ISH. (E) EBV-infected NPC tissue, included as a control, was obtained from the patients with GFAP-A in cohort B. CNS, central nervous system; EBV, Epstein-Barr virus; GFAP, glial fibrillar acidic protein; ISH, in situ hybridisation; NPC, nasopharyngeal carcinoma.