Autoantibody discovery across monogenic, acquired, and COVID19-associated autoimmunity with scalable PhIP-Seq

Abstract

Phage Immunoprecipitation-Sequencing (PhIP-Seq) allows for unbiased, proteome-wide autoantibody discovery across a variety of disease settings, with identification of disease-specific autoantigens providing new insight into previously poorly understood forms of immune dysregulation. Despite several successful implementations of PhIP-Seq for autoantigen discovery, including our previous work (Vazquez et al. 2020), current protocols are inherently difficult to scale to accommodate large cohorts of cases and importantly, healthy controls. Here, we develop and validate a high throughput extension of PhIP-seq in various etiologies of autoimmune and inflammatory diseases, including APS1, IPEX, RAG1/2 deficiency, Kawasaki Disease (KD), Multisystem Inflammatory Syndrome in Children (MIS-C), and finally, mild and severe forms of COVID19. We demonstrate that these scaled datasets enable machine-learning approaches that result in robust prediction of disease status, as well as the ability to detect both known and novel autoantigens, such as PDYN in APS1 patients, and intestinally expressed proteins BEST4 and BTNL8 in IPEX patients. Remarkably, BEST4 antibodies were also found in 2 patients with RAG1/2 deficiency, one of whom had very early onset IBD. Scaled PhIP-Seq examination of both MIS-C and KD demonstrated rare, overlapping antigens, including CGNL1, as well as several strongly enriched putative pneumonia-associated antigens in severe COVID19, including the endosomal protein EEA1. Together, scaled PhIP-Seq provides a valuable tool for broadly assessing both rare and common autoantigen overlap between autoimmune diseases of varying origins and etiologies.

Multi-omic longitudinal study reveals immune correlates of clinical course among hospitalized COVID-19 patients

“…For all VirScan libraries, the null distribution of each peptide’s log10 (rpK) was modeled using a set of 71 pre-pandemic healthy controls sera collected from the New York Blood Center. 137 Peptide counts were computed for all control samples, then were converted into proportions relative to the sum of sample counts. Each sample was downsampled by 1,500,000 weighted by the proportion values.…”

Pathogen and Antibody Identification in Children with Encephalitis in Myanmar

“…VirScan was performed in duplicate in accordance with the scaled, vacuum‐based, phage immunoprecipitation sequencing protocol, as described previously in detail 37 . Briefly, 10 μl of CSF was incubated overnight with 500 μl of phage library in pre‐blocked plates with continuous rocking.…”

“…Filtered lysate from round 1 was incubated with 10 μl of CSF for a second round. Library preparations were also performed as described, 37 using a Beckman acoustic (Echo 525) liquid handler for a 12.5‐μl reaction volume. Samples were sequenced on an Illumina NovaSeq 6000 for 147 base pair paired‐end sequencing.…”

“…VirScan was performed in duplicate in accordance with the scaled, vacuum-based, phage immunoprecipitation sequencing protocol, as described previously in detail. 37 Briefly, 10 μl of CSF was incubated overnight with 500 μl of phage library in pre-blocked Abbreviations: CT = computed tomography; IQR = interquartile range; LCL = lower control limit; MRI = magnetic resonance imaging; UCL = upper control limit.…”