Autoantibodies and cross-reactive immunity post SARS-CoV-2 infection
Creators: name: Moody, Rhiane
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 320404
subject: Cellular immunology
classificationCode: 320409
subject: Tumour immunology

description: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) is a novel human infecting virus that emerged in Wuhan, China in December 2019. Although primarily considered a respiratory disease, multiorgan pathologies have been reported, including autoimmune and autoimmune-like complications. As viral infections are known to be associated with autoimmunity, the aim of this thesis was to investigate IgG and IgM antibody responses to SARS-CoV-2 antigens and known autoantigens eight months post-COVID-19. Levels of these responses were further associated with self-reported initial clinical characteristics and outcomes at the time of sample collection. Additionally, the potential of molecular mimicry as a source of autoimmunity in COVID-19 was explored.

Plasma samples were collected from healthcare workers in Tasmania, Australia, eight months post an outbreak of COVID-19. This outbreak occurred in March 2020, early in the pandemic, and following the control of the outbreak, no community transmission in Tasmania occurred until December 2021. Given this, investigations of antibody responses were performed with either known COVID-19 negative or convalescent status post a singular infection, with no opportunity of additional infection prior to sample collection or vaccine intervention. 

First, IgG antibody responses to SARS-CoV-2 antigens (nucleoprotein, spike and spike subunits) and 102 human proteins with known autoimmune associations were measured and compared between the COVID-19 negative and COVID-19 convalescent cohorts.  COVID-19 convalescent individuals had significantly higher antibody responses to SARS-CoV-2 antigens highlighting lasting antibody responses up to eight months post infection. Additionally, on average the COVID-19 convalescent cohort had a higher number of positive autoantibody reactivities (5.1) in comparison to uninfected individuals (1.8), with the most common recognised autoantigens specific to the COVID-19 convalescent cohort. Of these, calprotectin, was the most common target, recognised by 22.6% of COVID-19 convalescent individuals. Furthermore, levels of anti-calprotectin were associated with feeling back to normal post infection. 

Next, levels of total immunoglobulin isotypes were measured, where total IgM was significantly increased in the COVID-19 convalescent cohort. IgM responses to SARS-CoV-2 antigen and other viral antigens were significant higher in the COVID-19 convalescent cohort in comparison to those not previously infected with SARS-CoV-2. In addition to increased IgM anti-viral responses, the COVID-19 convalescent cohort had significantly higher levels of antibody responses to 85% of the 102 autoantigens measured against. Levels of IgM antibody and autoantibody responses were not associated with self-reported clinical characteristics and outcomes. Strong positive correlations were found between responses to 62 autoantigens and SARS-CoV-2 antigens. Next, levels of cytokines and inflammatory biomarkers were measured with no differences found between the COVID-19 negative and convalescent cohorts. Within the COVID-19 convalescent cohort, the levels of 19 cytokines were associated with individuals who experienced a greater number of symptoms during the outbreak. 

Given the increased presence of IgG and IgM autoantibodies in the COVID-19 convalescent cohort, in silico techniques were used to investigate the potential of molecular mimicry as a mechanism of viral induced autoimmunity. Using the Immune Epitope Database (IEDB), linear B cell epitopes were predicted and selected for within several SARS-CoV-2 specific proteins. The 25 selected epitopes were interrogated for sequence homology to human proteins where 256 proteins shared sequence similarities with SARS-CoV-2 predicted epitopes. To investigate the potential of immune cross-reactivity between SARS-CoV-2 and human proteins, novel criteria that considers sequence similarity and alignment localisation was utilised. This identified B cell epitopes in human proteins with the potential of immune cross-reactivity with SARS-CoV-2 epitopes, not previously reported in other in silico studies which used direct sequence identity. 

Finally, we sought to validate predicted SARS-CoV-2 epitopes of interest from the in silico studies using the cohort of healthcare workers. IgG antibody responses were measured to 13 SARS-CoV-2 specific epitopes via ELISA (enzyme linked immunosorbent assay), 9 of which had at least one positive responder. Amongst the positive responders included individuals not previously infected with SARS-CoV-2, particularly with responses to nucleoprotein specific epitopes. Informatic tools were utilised to investigate the cause of these positive responses by comparing nucleoprotein epitopes with the commonly circulating human coronaviruses (HCoV). Of these only one epitope showed the potential to cross-react with one of the HCoVs. Given this, the nucleoprotein epitopes were interrogated for sequence similarities to non-coronavirus targets, which identified sequences from 9 proteins belonging to microbes found in the environment, human microbiome or human pathogens. However, an initial screening of responses to potential cross-reactive targets of one of the NP epitopes did not show cross-reactivity. 

This study furthers the knowledge of antibody and autoantibody responses post COVID-19 by identifying autoantigens recognised by IgG autoantibodies not previously highlighted in the literature, and the overall increase of IgM responses to a range of viral- and auto- antigens. It additionally provides insights of immune cross-reactivity in this disease.