Absence of specific autoantibodies in patients with narcolepsy type 1 as indicated by an unbiased random peptide-displayed phage screening

Abstract

Narcolepsy type 1 (NT1) is an enigmatic sleep disorder characterized by the selective loss of neurons producing orexin (also named hypocretin) in the lateral hypothalamus. Although NT1 is believed to be an autoimmune disease, the orexinergic neuron-specific antigens targeted by the pathogenic immune response remain elusive. In this study, we evaluated the differential binding capacity of various peptides to serum immunoglobin G from patients with NT1 and other hypersomnolence complaints (OHCs). These peptides were selected using an unbiased phage display technology or based on their significant presence in the serum of NT1 patients as identified from previous studies. Although the subtractive biopanning strategy successfully enriched phage clones with high reactivity against NT1 serum IgG, the 101 randomly selected individual phage clones could not differentiate the sera from NT1 and OHC. Compared to the OHC control group, serum from several NT1 patients exhibited increased reactivity to the 12-mer peptides derived from TRBV7, BCL-6, NRXN1, RXRG, HCRT, and RTN4 proteins, although not statistically significant. Collectively, employing both unbiased and targeted methodologies, we were unable to detect the presence of specific autoantibodies in our NT1 patient cohort. This further supports the hypothesis that the autoimmune response in NT1 patients likely stems primarily from T cell-mediated immunity rather than humoral immunity.

Fig 1. Schematic representation of the phage display panning procedure.

The positive selection (the 1st, 2nd, 3^rd, and 5th rounds) and negative selection (the 4th round) were performed against NT1 and OHC pooled serum IgG, respectively.

Fig 2. The persistence of phage output after each round of panning.

A. The white bars indicate the input number of random peptide-displayed phage particles in each round of panning, which is persistently kept at 3x10⁹ PFU. The black bars show phage particle titration at the ends of each round before they were amplified for the next round of input. B. Increased phage affinity against IgG OHC and NT1 pooled serum after each round of panning. The binding capacity of enriched phage pools to patient IgGs was measured by phage Elisa. The presenting data are mean ± SD (n = 2).

Fig 3. The binding of 101 phages with OHC and NT1 sera.

After five rounds of positive and negative biopanning, 101 randomly selected phage clones were evaluated for binding to IgG from pooled OHC and NT1 serum via phage ELISA (n = 3). The binding of phage with OHC pooled serum-derived and NT1 pooled serum-derived IgG are analyzed by multiple unpaired t-tests with p<0.05.

Fig 4. Binding of each phage clone toward individual serum IgG from 10 NT1 and 10 OHC.

Chosen phages were validated for specificity by indirect ELISA using individual IgG samples purified from OHC (n = 10) and NT1 serum (n = 10). Error bars indicate mean +/- SD, the paired t-test p values has been used to assess statistical significance.

Fig 5. Reactivity of serum antibody against the selected peptides assessed by ELISA.

Each dot represents an individual sample from the eight patients with other hypersomnolence complaints (OHC) and twenty-six patients with Narcolepsy type I (NT1). Multiple t-test p values are reported for the group comparison between OHC and NT1.