Exploring autoantigens in autoimmune limbic encephalitis using phage immunoprecipitation sequencing

To describe the use of high-throughput whole-human proteome phage immunoprecipitation sequencing (PhIP-Seq) in identifying potential antigens for antibody-negative autoimmune limbic encephalitis (ALE).

We used PhIP-Seq to analyze cerebrospinal fluid (CSF) from patients with antibody-negative ALE evaluated in our lab with detailed clinical records available (2008-2023). Identified autoantigens were validated using recombinant protein-based assay.

Of the 18 CSF samples from patients with ALE, 1 sample showed neuronal PAS domain protein 4 (NPAS4) as the putative autoantigen with the highest enrichment score. Patient presented with cognitive decline, gait dysfunction and unique MRI brain findings revealing asymmetric involvement of the medial temporal lobes with extension to the insular cortex and anterior temporal lobes. No malignancy was detected, and although the patient was initiated on immunotherapy, follow-up evaluation was limited. Another sample with very similar clinical and MRI brain findings, who developed ALE following COVID-19 infection, was referred for evaluation for neural-specific autoantibodies, and PhIP-Seq identified NPAS4 as the putative autoantigen. In both these samples, NPAS4-IgG was confirmed by cell-based assay (CBA) and enzyme-linked immunosorbent assay (ELISA). To assess the specificity of the NPAS4 autoantigen, a large cohort of disease (CSF, n = 49, serum, n = 220) and healthy controls (serum, n = 90) were tested by ELISA which were also negative. In addition, three samples had a high enrichment score for adenylate kinase 5 (AK5), an autoantigen associated with ALE. These patients also displayed a unique immunosignature that included AK5-IgG, which is distinct from NPAS4-IgG ALE or antibody-negative ALE. All three AK5-IgG seropositive cases presented with subacute memory loss, with MRI/PET brain florid showing medial temporal lobe involvement. No malignancy was detected in any of the three patients and immunotherapy led to improvement in one.

Our study demonstrates the utility of high-throughput PhIP-Seq in identifying potential autoantigens in antibody-negative ALE. The identification of NPAS4 as a putative antigen and identification of additional AK5-IgG-positive cases, underscores the potential of PhIP-Seq as a powerful tool in autoimmune disease research.