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\begin{document}

\title{Establishing the prevalence of common, clinically relevant
tissue-specific autoantibodies following SARS CoV-2 infection}



\author[1]{A. G. Richter}%
\author[1]{Adrian Shields}%
\author[1]{Abid Karim}%
\author[1]{David Birch}%
\author[1]{Sian Faustini}%
\author[1]{Lora Steadman}%
\author[1]{Kerensa Ward}%
\author[1]{Tim Plant}%
\author[2]{Gary Reynolds}%
\author[3]{Tonny Veenith}%
\author[4]{A Cunningham}%
\author[5]{M Drayson}%
\author[1]{David Wraith}%
\affil[1]{University of Birmingham}%
\affil[2]{NIHR}%
\affil[3]{University Hospitals Birmingham NHS Foundation Trust}%
\affil[4]{Birmingham University Medical School}%
\affil[5]{University of Birmingham Medical School}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
COVID-19 has been associated with both transient and persistent systemic
symptoms that do not appear to be a direct consequence of viral
infection. The generation of autoantibodies has been proposed as a
mechanism to explain these symptoms. To understand this phenomenon in
more detail, we investigated the frequency and specificity of clinically
relevant autoantibodies in 84 individuals previously infected with
SARS-CoV-2, suffering from COVID-19 of varying severity in both the
acute and convalescent setting. These were compared with results from 32
individuals who were on ITU for non COVID reasons. We demonstrate a
higher frequency of autoantibodies in the COVID-19 ITU group compared
with non-COVID-19 ITU disease control patients and that autoantibodies
were also found in the serum 3-5 months post COVID-19 infection.
Non-COVID patients displayed a diverse pattern of autoantibodies; in
contrast, the COVID-19 groups had a more restricted panel of
autoantibodies including skin, skeletal muscle and cardiac antibodies.
Our results demonstrate that severe COVID-19 induces a pattern of
autoantibodies that may correlate with and contribute to the immune
pathology associated with the long-term sequelae of infection.%
\end{abstract}%



\sloppy


\emph{Establishing the prevalence of common, clinically relevant
tissue-specific autoantibodies following SARS CoV-2 infection}

Alex G. Richter\textsuperscript{1} , Adrian M.
Shields\textsuperscript{1}, Abid Karim\textsuperscript{1}, David
Birch\textsuperscript{1}, Sian E. Faustini\textsuperscript{1}, Lora
Steadman\textsuperscript{2}, Kerensa Ward \textsuperscript{1}, Timothy
Plant\textsuperscript{1}, Gary Reynolds\textsuperscript{2}, Tonny
Veenith\textsuperscript{3}, Adam F. Cunningham\textsuperscript{2}, Mark
T. Drayson\textsuperscript{1}, David C. Wraith\textsuperscript{2}

Clinical Immunology Service, Institute for Immunology and Immunotherapy,
University of Birmingham, Birmingham, UK

Institute of Immunology and Immunotherapy, University of Birmingham, UK

Department of Critical Care Medicine, University Hospitals Birmingham
NHS Trust, Birmingham, UK

Key words: COVID-19, SARS-CoV-2, autoimmunity, autoantibodies, Long
COVID

Word count:

Number of figures: 2

Number of tables: 2

Supplementary data: 2 tables

\textbf{Correspondence to:}

Professor Alex Richter

Clinical Immunology Service

Institute for Immunology and Immunotherapy

University of Birmingham

Birmingham, UK

a.g.richter@bham.ac.uk

\textbf{Abstract}

COVID-19 has been associated with both transient and~persistent~systemic
symptoms that do not appear to be~a direct consequence of viral
infection. ~The generation of autoantibodies has been proposed as a
mechanism to explain these symptoms. To understand this phenomenon in
more detail,~we investigated the frequency and specificity of clinically
relevant autoantibodies in 84 individuals previously infected with
SARS-CoV-2, suffering from COVID-19 of varying severity in both the
acute and convalescent setting.~ These were compared with results from
32 individuals who were on ITU for non COVID reasons.

We demonstrate~a higher frequency of autoantibodies in the COVID-19 ITU
group compared with non-COVID-19 ITU disease control patients~and that
autoantibodies were also found in the~serum~3-5 months post COVID-19
infection.~Non-COVID patients displayed a diverse pattern of
autoantibodies; in contrast, the COVID-19 groups had a more restricted
panel of autoantibodies including skin, skeletal muscle and cardiac
antibodies. ~ Our results demonstrate that severe COVID-19 induces a
pattern of autoantibodies that may correlate with and contribute to the
immune pathology associated with the long-term sequelae of infection.



\textbf{Introduction}

Infection is a common event that can disrupt immunological tolerance
and, in some circumstances, lead to autoimmune disease {[}1{]}.Viral
infections have been linked to both the initiation of a range of
autoimmune diseases and disease relapse in individuals with existing
conditions {[}2{]}. For most autoimmune diseases, it is not clear
whether infection is the sole precipitating event, an inevitable
consequence of a genetic predisposition or whether infection is a
necessary trigger in a genetically susceptible individual.

Early data suggest that autoimmune phenomena may exacerbate the immune
pathology associated with SARS-CoV-2 infection or trigger long-term
autoimmune complications secondary to bystander activation or molecular
mimicry. There are reports of SARS-CoV-2 infection being associated with
a number of autoimmune disorders including Guillain-Barre Syndrome (GBS)
{[}3{]}and various cytopenias {[}4{]}. Anti-phospholipid antibodies have
been detected in \textasciitilde{}50\% of hospitalised patients and
linked to an increased incidence of cerebral infarction; however, the
clinical relevance of this observation in COVID-19 remains controversial
as anti-phospholipid antibody generation in acute illness is a common,
non-specific finding {[}5-7{]}. Also, neutralising antibodies against
type 1 anti-viral cytokines, Interferon (IFN)-\selectlanguage{greek}ω \selectlanguage{english}and/or IFN-\selectlanguage{greek}α \selectlanguage{english}have been
found in over 10\% of patients with COVID-19 pneumonia {[}8{]}. By
screening a yeast expression library, Wang et al. identified
autoantibodies against cytokines (including type 1 IFNs), CNS antigens
and extracellular matrix proteins whose frequency correlated with
disease severity {[}9{]}.

Paediatric multisystem Inflammatory Syndrome (PIMS-TS) is a rare
condition that occurs as a late complication of SARS-CoV-2 infection.
Children suffering from this post-COVID inflammatory condition were also
seropositive for anti-endothelial antibodies that may contribute to
their pathology {[}10-12{]}. We and others are now searching for tissue
specific autoantibodies in adults. Kreye and colleagues screened for CNS
autoantibodies using murine brain sections identifying anti-Yo and
NMDA-R as well as a variety of antibodies against epitopes including
vessel endothelium {[}12{]}. Evidence has arisen that some of these
antibodies may arise through cross-reactive recognition of self-antigens
by antibodies specific for SARS-CoV-2{[}13{]}.

Identifying the relationship between autoimmune antibody induction and
COVID-19 is further complicated by the spectrum of presentations of this
disease. In particular, the severity of disease may require
hospitalisation and the more severe presentations of disease may
influence the maintenance of autoantibodies during convalescence. To
investigate any potential links between SARS-CoV-2 infection and
autoantibodies we examined sera from acute and convalescent COVID-19
patients, some of whom had been hospitalised, for the presence of
autoantibodies to a spectrum of antigens by indirect immunofluorescence.
We identify a high frequency and wide range of clinically relevant
autoantibodies in both acute and convalescent samples from COVID-19
patients. Their frequency and tissue specificity suggests that
autoantibodies may contribute to the long-term consequences of COVID-19.



\textbf{Methods}

Participants Four cohorts of participants were recruited: the first
group included 24 convalescent health care workers who had suffered from
COVID-19 and not required hospitalisation to determine whether disease
severity makes a difference to the generation of any autoantibodies. The
second group included 25 individuals who were currently on the intensive
therapy unit (ITU) diagnosed with COVID-19. The third group included 32
individuals who were currently on the ITU but for a reason other than
COVID-19 to determine whether acute illness per se was associated with
autoantibody production. The fourth group included 35 individuals who
had been admitted to ITU with COVID-19 and had been discharged and their
samples were taken at 3-6 month review. This group explored persistence
of any potential antibodies.

Autoantibody assays Autoantibodies were detected on serum samples using
indirect immunofluorescence. A spectrum of anti-neutrophil and organ
specific autoantibodies were tested as a broad screen using standard
ISO15189 accredited clinical tests. The immunoglobulin isotype detected
was IgG with the exception of endomysial antibodies which are IgA. The
full list of assays, manufacturers and disease association are described
in the\textbf{Supplementary data} but in short we undertook indirect
immunofluorescence on commercial pre-prepared slides to detect IgG
antibodies for adrenal, autoimmune encephalitis, anti-neutrophil (ANA),
anti-neutrophil cytoplasmic ANCA, cardiac, epidermal, islet cell, a
range of cerebellar (Purkinje cell) antibodies, smooth muscle,
mitochondrial, gastric parietal cell and skeletal muscle antibodies and
IgA endomysial antibodies. All samples were read by two experienced
clinical laboratory scientists for agreement. Data are largely
descriptive. Inter group comparison was made by Chi Square using
GraphPad Prism 9.

Ethical approval

For the acute cohort of ITU patient, surplus anonymised samples from
routine clinical testing were used and for the convalescent ITU patients
they were consented in clinic; ethical approval for these groups was
granted by the North West-Preston Research Committee (ref 20/NW/0240
IRAS Project ID: 282164). The health care worker cohort was a random
subgroup of COVID antibody positive patients from the COvid-19
COnvalescent immunity study (COCO) study approved by the London - Camden
\& Kings Cross Research Ethics Committee reference 20/HRA/1817.



\textbf{Results}

There were 116 patient samples tested; 32 from group 1 (acute,
non-COVID-19, ITU) 25 from group 2 (acute, COVID-19, ITU), 35 from group
3 (convalescent, COVID-19, post ITU) and 24 from group 4 (convalescent,
COVID-19, non-hospitalised). The demographic descriptions
in\textbf{Table 1} find a female preponderance and slightly younger
cohort in the non-hospitalised convalescent group. The average time from
symptom onset was longer in the post ITU patients (151 days) than the
non-hospitalised convalescent cohort (38 days). Non-white ethnicity is
higher in all the COVID groups than the non-COVID ITU cohort, in keeping
with known risk factors for severe COVID {[}14{]}. The details of number
of samples tested are described in \textbf{Table 2} ; for some patients
there was insufficient sample to run all tests.

The number of autoantibodies varied between groups. The highest numbers
of autoantibodies to different antigenic targets was detected in the
severe COVID disease groups. In group 1 (acute Non COVID-19, ITU) 13/32
(41\%) individuals had autoantibodies; 8 tested positive for 1
autoantibody, 4 for 2 autoantibodies and 1 for 3. For group 2 (acute
COVID-19, ITU) 15/25 (60\%) individuals had autoantibodies; 12 tested
positive for 1 autoantibody, 1 for 2 autoantibodies and 2 for 3. In
group 3 (convalescent COVID-19, post ITU) 27/36 (75\%) individuals had
autoantibodies, 14 tested positive for 1 autoantibody, 10 for 2
autoantibodies and 3 for 3. In group 4 (convalescent, COVID-19,
non-hospitalised) 13/24 (54\%) individuals had autoantibodies and none
tested positive for more than one (\textbf{Figure 1} ).

Representative slides of epidermal, skeletal, cardiac and smooth muscle
autoantibodies are shown in \textbf{Figure 2} . In the acute Non
COVID-19, ITU patients there were many different causes of their illness
(Supplementary Table 1) and autoantibodies were found against nearly all
(12/13) of the different autoantigens examined, indicating a more random
distribution. A higher proportion of acute COVID-19, ITU patients had
autoantibodies (60\% versus 41\%) but to a narrower range of
autoantigens (7/13) with a preponderance of epidermal (41\%) and
skeletal antibodies (17\%). This preponderance was seen in convalescent,
COVID-19, post ITU with epidermal (19\%) and skeletal antibodies (19\%)
but additionally cardiac muscle antibodies (28\%) and smooth muscle
antibodies (31\%). Representative slides of epidermal, smooth muscle,
skeletal muscle and cardiac muscle autoantibodies are shown in

\textbf{Figure 2.}

In the convalescent, COVID-19, non-hospitalised cohort fewer individuals
had autoantibodies than the convalescent ITU cohort (54\% versus 75\%)
and against only four autoantigens: epidermal (25\%), smooth muscle
(17\%), ANCA (8\%) and gastric parietal (4\%). These results suggest
that COVID-19 infection is associated with autoantibody induction, and
that these antibodies target a limited repertoire of self-antigens.

\textbf{Discussion}

SARS-CoV-2 is associated with a spectrum of illness during the acute
viral infection, persisting during convalescence and as part of the long
COVID syndrome. Constitutional, respiratory, cardiac, neurological,
musculoskeletal and psychiatric symptoms are being increasingly
described but the mechanisms behind these are uncertain {[}15{]}. It is
not known whether these phenomena arise as direct effect of the virus or
from off-target immune effects, including autoimmunity. Our study finds
that there is a high prevalence of autoantibodies found in the acute and
convalescent phase of COVID-19 suggesting that SARS-CoV-2 infection is
associated with significant perturbations of immunological tolerance and
raising the possibility that autoimmunity may play a role in the
pathogenesis of acute and chronic symptoms.

Two fifths of the acute non COVID-19 ITU patients had autoantibodies
suggesting that acute severe illness per se is associated with
autoantibody production and the wide range of target autoantigens may
reflect the diversity of this cohorts' underlying disease. Three fifths
of the acute COVID-19, ITU patients had autoantibodies and these were of
a narrower diversity with antibodies against epidermal intercellular
cement and skeletal muscle predominant. These antibodies were persistent
over time and also detected in convalescence post ITU COVID-19, over 5
months from symptoms onset. In addition, cardiac and smooth muscle
antibodies were also identified. Cardiac and skeletal muscle
autoantibodies were not found in convalescent individuals with
non-hospitalised COVID-19, although smooth muscle antibodies were
detected and a quarter had antibodies directed at epidermal
intercellular cement. The narrow array of tissue specific autoantibodies
associated with COVID-19 infection suggests loss of tolerance to these
antigens may be disease specific, rather than a phenomenon associated
with critical illness.

The link between infection and autoimmunity is well described with
multiple genetic and environmental factors implicated {[}1{]}.
Pathogenic mechanisms elucidated include molecular mimicry, epitope
spreading, revelation of cryptic antigen and bystander activation,
although which specific mechanism in which situation is usually
uncertain. Similarly, just because an autoantibody is generated doesn't
necessarily mean that the autoantibody is pathogenic. In some conditions
such as myasthenia gravis there is a clear link between acetyl choline
receptor antibodies and dysfunction of the motor end plate whereas in
some conditions such as systemic lupus erythematosus the presence of
high titre ANA is a non-specific biomarker of disease and the
autoantibodies are not thought to be pathogenic. One of the limitations
in understanding the role of autoantibodies in infectious disease has
been the relative paucity of cases that are available to study within a
reasonable time-frame. The sheer extent of the COVID-19 pandemic
obviously overcomes this and work such as that presented here are first
steps in interrogating these links.

The pattern of skin and muscle autoantibodies is intriguing and further
studies are needed to elucidate the antigenic target and the clinical
significance of these autoantibodies. One interesting possibility is the
desmoglein family and DSG1 and 3 are found in the autoimmune blistering
pemphigus disorders. Whilst oral ulceration and blistering has been
described in COVID-19 {[}16, 17{]}) it is by no means a commonly
reported feature in large clinical studies such as ISARIC4C (Coronavirus
Clinical Characterisation Consortium) {[}18{]}.

This is an observational hypothesis generating clinical laboratory study
that has raised the possibility of COVID infection generating a specific
spectrum of autoantibodies. Determining the clinical relevance of these
findings is still required but highlights specific history and
examination that should be undertaken in COVID follow up clinics.
Further studies examining the link between autoantibodies and COVID-19
are required to help understand how one virus can drive a myriad of
presentations in different patients.

\textbf{Acknowledgements}

The authors would like to acknowledge the staff of the University of
Birmingham Clinical Immunology Service for facilitating laboratory
studies. The convalescent health care worker study COCO was carried out
at the National Institute for Health Research (NIHR)/Wellcome Trust
Birmingham Clinical Research Facility.

This study was funded as part of the UK Coronavirus Immunology
Consortium funded by NIHR and UKRI. This paper presents independent
research supported by the NIHR Birmingham Biomedical Research Centre at
the University Hospitals Birmingham NHS Foundation Trust and the
University of Birmingham.

\textbf{Competing interests}

MTD reports personal fees from Abingdon Health, outside the submitted
work. All other authors declare no competing interests.

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